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1
Smith, Jason A., Laura A. Bannister, Vikram Bhattacharjee, Yibin Wang, Barbara Criscuolo Waldman, and Alan S. Waldman. "Accurate Homologous Recombination Is a Prominent Double-Strand Break Repair Pathway in Mammalian Chromosomes and Is Modulated by Mismatch Repair Protein Msh2." Molecular and Cellular Biology 27, no. 22 (September 10, 2007): 7816–27. http://dx.doi.org/10.1128/mcb.00455-07.
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ABSTRACT We designed DNA substrates to study intrachromosomal recombination in mammalian chromosomes. Each substrate contains a thymidine kinase (tk) gene fused to a neomycin resistance (neo) gene. The fusion gene is disrupted by an oligonucleotide containing the 18-bp recognition site for endonuclease I-SceI. Substrates also contain a “donor” tk sequence that displays 1% or 19% sequence divergence relative to the tk portion of the fusion gene. Each donor serves as a potential recombination partner for the fusion gene. After stably transfecting substrates into mammalian cell lines, we investigated spontaneous recombination and double-strand break (DSB)-induced recombination following I-SceI expression. No recombination events between sequences with 19% divergence were recovered. Strikingly, even though no selection for accurate repair was imposed, accurate conservative homologous recombination was the predominant DSB repair event recovered from rodent and human cell lines transfected with the substrate containing sequences displaying 1% divergence. Our work is the first unequivocal demonstration that homologous recombination can serve as a major DSB repair pathway in mammalian chromosomes. We also found that Msh2 can modulate homologous recombination in that Msh2 deficiency promoted discontinuity and increased length of gene conversion tracts and brought about a severalfold increase in the overall frequency of DSB-induced recombination.
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Aoki, Tatsuhiko, Hiroaki Kariyazaki, Koji Sueoka, and Kouji Izunome. "Gettering Efficiency of Si (110)/(100) Directly Bonded Hybrid Crystal Orientation Substrates." Solid State Phenomena 156-158 (October 2009): 369–73. http://dx.doi.org/10.4028/www.scientific.net/ssp.156-158.369.
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We have investigated the gettering efficiency at the interface of Si (110) and Si (100) directly bonded (DSB) substrates. DSB substrates were prepared by conventional bonding and grinding back methods. DSB substrates were intentionally contaminated with 3d transition metals (Fe, Ni, Cu) and then annealed at 1000 oC. The dependence of metal concentrations on the depth was evaluated by a secondary ionization mass spectrometer (SIMS). Furthermore, we observed the interface of DSB by transmission electron microscope (TEM), and characterized the form of the gettered metals.
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Lin, Yunfu, Tamas Lukacsovich, and Alan S. Waldman. "Multiple Pathways for Repair of DNA Double-Strand Breaks in Mammalian Chromosomes." Molecular and Cellular Biology 19, no. 12 (December 1, 1999): 8353–60. http://dx.doi.org/10.1128/mcb.19.12.8353.
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ABSTRACT To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.
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Kato, Tetsuji, Yuji Ohara, Takaya Ueda, Jun Kikkawa, Yoshiaki Nakamura, Akira Sakai, Osamu Nakatsuka, et al. "Microscopic Structure of Directly Bonded Silicon Substrates." Key Engineering Materials 470 (February 2011): 164–70. http://dx.doi.org/10.4028/www.scientific.net/kem.470.164.
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Using X-ray microdiffraction (XRMD) and transmission electron microscopy (TEM) techniques, we have investigated the microscopic structure of Si(011)/Si(001) direct silicon bonding (DSB) substrates. XRMD was performed to measure the local lattice spacing and tilting in the samples before and after oxide out-diffusion annealing. Diffraction analyses for (022) lattice planes with two orthogonal in-plane directions of X-ray incidence revealed anisotropic domain textures in the Si(011) layer. Such anisotropy was also confirmed by TEM in the morphology at the Si(011)/Si(001) bonded interface. The anisotropic crystallinity is discussed on the basis of interfacial defect structures which are proper to the DSB substrate.
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Dronkert, Mies L. G., H. Berna Beverloo, Roger D. Johnson, Jan H. J. Hoeijmakers, Maria Jasin, and Roland Kanaar. "Mouse RAD54 Affects DNA Double-Strand Break Repair and Sister Chromatid Exchange." Molecular and Cellular Biology 20, no. 9 (May 1, 2000): 3147–56. http://dx.doi.org/10.1128/mcb.20.9.3147-3156.2000.
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ABSTRACT Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54wild-type and knockout cells revealed direct evidence for a role ofmRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence ofmRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.
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Osman, Fekret, Elizabeth A. Fortunato, and Suresh Subramani. "Double-Strand Break-Induced Mitotic Intrachromosomal Recombination in the Fission Yeast Schizosaccharomyces pombe." Genetics 142, no. 2 (February 1, 1996): 341–57. http://dx.doi.org/10.1093/genetics/142.2.341.
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Abstract The Saccharomyces cerevisiae HO gene and MATa cutting site were used to introduce site-specific double-strand breaks (DSBs) within intrachromosomal recombination substrates in Schizosaccharomyces pombe. The recombination substrates consisted of nontandem direct repeats of ade6 heteroalleles. DSB induction stimulated the frequency of recombinants 2000-fold. The spectrum of DSB-induced recombinants depended on whether the DSB was introduced within one of the ade6 repeats or in intervening unique DNA. When the DSB was introduced within unique DNA, over 99.8% of the recombinants lacked the intervening DNA but retained one copy of ade6 that was wild type or either one of the heteroalleles. When the DSB was located in duplicated DNA, 77% of the recombinants were similar to the deletion types described above, but the single ade6 copy was either wild type or exclusively that of the uncut repeat. The remaining 23% of the induced recombinants were gene convertants with two copies of ade6 and the intervening sequences; the ade6 heteroallele in which the DSB was induced was the recipient of genetic information. Half-sectored colonies were isolated, analyzed and interpreted as evidence of heteroduplex DNA formation. The results are discussed in terms of current models for recombination.
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Nussbaum, A., M. Shalit, and A. Cohen. "Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli." Genetics 130, no. 1 (January 1, 1992): 37–49. http://dx.doi.org/10.1093/genetics/130.1.37.
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Abstract To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI+ cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break-repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction.
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Rattray, Alison J., Brenda K. Shafer, Carolyn B. McGill, and Jeffrey N. Strathern. "The Roles of REV3 and RAD57 in Double-Strand-Break-Repair-Induced Mutagenesis of Saccharomyces cerevisiae." Genetics 162, no. 3 (November 1, 2002): 1063–77. http://dx.doi.org/10.1093/genetics/162.3.1063.
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Abstract The DNA synthesis associated with recombinational repair of chromosomal double-strand breaks (DSBs) has a lower fidelity than normal replicative DNA synthesis. Here, we use an inverted-repeat substrate to monitor the fidelity of repair of a site-specific DSB. DSB induction made by the HO endonuclease stimulates recombination >5000-fold and is associated with a >1000-fold increase in mutagenesis of an adjacent gene. We demonstrate that most break-repair-induced mutations (BRIMs) are point mutations and have a higher proportion of frameshifts than do spontaneous mutations of the same substrate. Although the REV3 translesion DNA polymerase is not required for recombination, it introduces ∼75% of the BRIMs and ∼90% of the base substitution mutations. Recombinational repair of the DSB is strongly dependent upon genes of the RAD52 epistasis group; however, the residual recombinants present in rad57 mutants are associated with a 5- to 20-fold increase in BRIMs. The spectrum of mutations in rad57 mutants is similar to that seen in the wild-type strain and is similarly affected by REV3. We also find that REV3 is required for the repair of MMS-induced lesions when recombinational repair is compromised. Our data suggest that Rad55p/Rad57p help limit the generation of substrates that require pol ζ during recombination.
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González-Barrera, Sergio, María García-Rubio, and Andrés Aguilera. "Transcription and Double-Strand Breaks Induce Similar Mitotic Recombination Events inSaccharomyces cerevisiae." Genetics 162, no. 2 (October 1, 2002): 603–14. http://dx.doi.org/10.1093/genetics/162.2.603.
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AbstractWe have made a comparative analysis of double-strand-break (DSB)-induced recombination and spontaneous recombination under low- and high-transcription conditions in yeast. We constructed two different recombination substrates, one for the analysis of intermolecular gene conversions and the other for intramolecular gene conversions and inversions. Such substrates were based on the same leu2-HOr allele fused to the tet promoter and containing a 21-bp HO site. Gene conversions and inversions were differently affected by rad1, rad51, rad52, and rad59 single and double mutations, consistent with the actual view that such events occur by different recombination mechanisms. However, the effect of each mutation on each type of recombination event was the same, whether associated with transcription or induced by the HO-mediated DSB. Both the highly transcribed DNA and the HO-cut sequence acted as recipients of the gene conversion events. These results are consistent with the hypothesis that transcription promotes initiation of recombination along the DNA sequence being transcribed. The similarity between transcription-associated and DSB-induced recombination suggests that transcription promotes DNA breaks.
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Willers, Henning, Fen Xia, and Simon N. Powell. "Recombinational DNA Repair in Cancer and Normal Cells: The Challenge of Functional Analysis." Journal of Biomedicine and Biotechnology 2, no. 2 (2002): 86–93. http://dx.doi.org/10.1155/s1110724302204027.
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A major goal of current cancer research is to understand the functional consequences of mutations in recombinational DNA repair genes. The introduction of artificial recombination substrates into living cells has evolved into a powerful tool to perform functional analysis of DNA double strand break (DSB) repair. Here, we review the principles and practice of current plasmid assays with regard to the two major DSB repair pathways, homologous recombination and nonhomologous end-joining. A spectrum of assay types is available to assess repair in a wide variety of cell lines. However, several technical challenges still need to be overcome. Understanding the alterations of DSB repair in cancers will ultimately provide a rational basis for drug design that may selectively sensitize tumor cells to ionizing radiation and chemotherapy, thereby achieving therapeutic gain.
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Camargo, Edwaldo E., Maria K. Sato, Gilda M. B. Del Negro, and Carlos da Silva Lacaz. "Radiometric detection of metabolic activity of Paracoccidioides brasiliensis and its susceptibility to amphotericin B and diethylstilbestrol." Revista do Instituto de Medicina Tropical de São Paulo 29, no. 5 (October 1987): 289–94. http://dx.doi.org/10.1590/s0036-46651987000500005.
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Paracoccidioidomycosis (South American blastomycosis) is a systemic disease, strikingly more frequent in males, caused by the dimorphic fungus Paracoccidioides brasiliensis. A radiometric assay system has been applied to study the metabolic activity and the effect of drugs on this fungus "in vitro". The Y form of the yeast, grown in liquid Sabouraud medium was inoculated into sterile reaction vials containing the 6B aerobic medium along with 2.0 μCi of 14C-substrates. Control vials, prepared in the same way, contained autoclaved fungi. To study the effects of amphotericin B (AB) (0.1 and 10 μg/ml) and diethylstilbestrol (DSB) (1.0, 5.0 and 10 μg/ml) extra controls with live fungi and no drug were used. All vials were incubated at 35°C and metabolism measured daily with a Bactec instrument. 14CO2 production by P. brasiliensis was slow and could be followed for as long as 50 days. AB at 10mg/ml and DSB at 5 μg/ml inhibited the metabolism and had a cidal effect on this fungus. The results with DSB might explain the low incidence of the disease in females. This technique shows promise for studying metabolic pathways, investi gating more convenient 14C-substrates to expedite radiometric detection and for monitoring the effects of other drugs and factors on the metabolism of P. brasiliensis "in vitro".
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Kang, Jian, David Ferguson, Hoseok Song, Craig Bassing, Mark Eckersdorff, Frederick W. Alt, and Yang Xu. "Functional Interaction of H2AX, NBS1, and p53 in ATM-Dependent DNA Damage Responses and Tumor Suppression." Molecular and Cellular Biology 25, no. 2 (January 15, 2005): 661–70. http://dx.doi.org/10.1128/mcb.25.2.661-670.2005.
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ABSTRACT Ataxia-telangiectasia (A-T) mutated (ATM) kinase signals all three cell cycle checkpoints after DNA double-stranded break (DSB) damage. H2AX, NBS1, and p53 are substrates of ATM kinase and are involved in ATM-dependent DNA damage responses. We show here that H2AX is dispensable for the activation of ATM and p53 responses after DNA DSB damage. Therefore, H2AX functions primarily as a downstream mediator of ATM functions in the parallel pathway of p53. NBS1 appears to function both as an activator of ATM and as an adapter to mediate ATM activities after DNA DSB damage. Phosphorylation of ATM and H2AX induced by DNA DSB damage is normal in NBS1 mutant/mutant (NBS1m/m) mice that express an N-terminally truncated NBS1 at lower levels. Therefore, the pleiotropic A-T-related systemic and cellular defects observed in NBS1m/m mice are due to the disruption of the adapter function of NBS1 in mediating ATM activities. While H2AX is required for the irradiation-induced focus formation of NBS1, our findings indicate that NBS1 and H2AX have distinct roles in DNA damage responses. ATM-dependent phosphorylation of p53 and p53 responses are largely normal in NBS1m/m mice after DNA DSB damage, and p53 deficiency greatly facilitates tumorigenesis in NBS1m/m mice. Therefore, NBS1, H2AX, and p53 play synergistic roles in ATM-dependent DNA damage responses and tumor suppression.
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Schildkraut, Ezra, Cheryl A. Miller, and Jac A. Nickoloff. "Transcription of a Donor Enhances Its Use during Double-Strand Break-Induced Gene Conversion in Human Cells." Molecular and Cellular Biology 26, no. 8 (April 15, 2006): 3098–105. http://dx.doi.org/10.1128/mcb.26.8.3098-3105.2006.
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ABSTRACT Homologous recombination (HR) mediates accurate repair of double-strand breaks (DSBs) but carries the risk of large-scale genetic change, including loss of heterozygosity, deletions, inversions, and translocations. Nearly one-third of the human genome consists of repetitive sequences, and DSB repair by HR often requires choices among several homologous repair templates, including homologous chromosomes, sister chromatids, and linked or unlinked repeats. Donor preference during DSB-induced gene conversion was analyzed by using several HR substrates with three copies of neo targeted to a human chromosome. Repair of I-SceI nuclease-induced DSBs in one neo (the recipient) required a choice between two donor neo genes. When both donors were downstream, there was no significant bias for proximal or distal donors. When donors flanked the recipient, we observed a marked (85%) preference for the downstream donor. Reversing the HR substrate in the chromosome eliminated this preference, indicating that donor choice is influenced by factors extrinsic to the HR substrate. Prior indirect evidence suggested that transcription might increase donor use. We tested this question directly and found that increased transcription of a donor enhances its use during gene conversion. A preference for transcribed donors would minimize the use of nontranscribed (i.e., pseudogene) templates during repair and thus help maintain genome stability.
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Sugawara, Neal, Grzegorz Ira, and James E. Haber. "DNA Length Dependence of the Single-Strand Annealing Pathway and the Role of Saccharomyces cerevisiae RAD59 in Double-Strand Break Repair." Molecular and Cellular Biology 20, no. 14 (July 15, 2000): 5300–5309. http://dx.doi.org/10.1128/mcb.20.14.5300-5309.2000.
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ABSTRACT A DNA double-strand break (DSB) created by the HO endonuclease inSaccharomyces cerevisiae will stimulate recombination between flanking repeats by the single-strand annealing (SSA) pathway, producing a deletion. Previously the efficiency of SSA, using homologous sequences of different lengths, was measured in competition with that of a larger repeat further from the DSB, which ensured that nearly all cells would survive the DSB if the smaller region was not used (N. Sugawara and J. E. Haber, Mol. Cell. Biol. 12:563–575, 1992). Without competition, the efficiency with which homologous segments of 63 to 205 bp engaged in SSA was significantly increased. A sequence as small as 29 bp was used 0.2% of the time, and homology dependence was approximately linear up to 415 bp, at which size almost all cells survived. A mutant with a deletion of RAD59, a homologue of RAD52, was defective for SSA, especially when the homologous-sequence length was short; however, even with 1.17-kb substrates, SSA was reduced fourfold. DSB-induced gene conversion also showed a partial dependence on Rad59p, again being greatest when the homologous-sequence length was short. We found that Rad59p plays a role in removing nonhomologous sequences from the ends of single-stranded DNA when it invades a homologous DNA template, in a manner similar to that previously seen with srs2 mutants. Δrad59 affected DSB-induced gene conversion differently from msh3 and msh2, which are also defective in removing nonhomologous ends in both DSB-induced gene conversion and SSA. A msh3 rad59 double mutant was more severely defective in SSA than either single mutant.
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He, Deyun, Zhen Du, Huiling Xu, and Xiaoming Bao. "Chl1, an ATP-Dependent DNA Helicase, Inhibits DNA:RNA Hybrids Formation at DSB Sites to Maintain Genome Stability in S. pombe." International Journal of Molecular Sciences 23, no. 12 (June 14, 2022): 6631. http://dx.doi.org/10.3390/ijms23126631.
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As an ATP-dependent DNA helicase, human ChlR1/DDX11 (Chl1 in yeast) can unwind both DNA:RNA and DNA:DNA substrates in vitro. Studies have demonstrated that ChlR1 plays a vital role in preserving genome stability by participating in DNA repair and sister chromatid cohesion, whereas the ways in which the biochemical features of ChlR1 function in DNA metabolism are not well understood. Here, we illustrate that Chl1 localizes to double-strand DNA break (DSB) sites and restrains DNA:RNA hybrid accumulation at these loci. Mutation of Chl1 strongly impairs DSB repair capacity by homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways, and deleting RNase H further reduces DNA repair efficiency, which indicates that the enzymatic activities of Chl1 are needed in Schizosaccharomyces pombe. In addition, we found that the Rpc37 subunit of RNA polymerase III (RNA Pol III) interacts directly with Chl1 and that deletion of Chl1 has no influence on the localization of Rpc37 at DSB site, implying the role of Rpc37 in the recruitment of Chl1 to this site.
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Elliott, Beth, and Maria Jasin. "Repair of Double-Strand Breaks by Homologous Recombination in Mismatch Repair-Defective Mammalian Cells." Molecular and Cellular Biology 21, no. 8 (April 15, 2001): 2671–82. http://dx.doi.org/10.1128/mcb.21.8.2671-2682.2001.
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ABSTRACT Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93–101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2 −/− cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.
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Kato, Tetsuji, Takaya Ueda, Yuji Ohara, Jun Kikkawa, Yoshiaki Nakamura, Akira Sakai, Osamu Nakatsuka, et al. "Structural Change during the Formation of Directly Bonded Silicon Substrates." Key Engineering Materials 470 (February 2011): 158–63. http://dx.doi.org/10.4028/www.scientific.net/kem.470.158.
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The use of Si(011)/Si(001) direct silicon bonding (DSB) substrates is a key element of future complementary metal-oxide-semiconductor device technology. In the conventional bonding process, it is necessary to remove interfacial SiO2 to achieve direct atomic bonding. In this study, using X-ray microdiffraction and transmission electron microscopy, we investigate the structural changes caused by oxide out-diffusion annealing (ODA). It is revealed that crystallinity of the bonded Si(011) layer is degraded after low temperature ODA and gradually recovered with an increase in the ODA temperature and annealing time, which is well correlated with the interfacial SiO2/Si morphology. Characteristic domain textures depending on the ODA temperature are also detected.
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Muyrers, Joep P. P., Youming Zhang, Fraenk Buchholz, and A. Francis Stewart. "RecE/RecT and Redα/Redβ initiate double-stranded break repair by specifically interacting with their respective partners." Genes & Development 14, no. 15 (August 1, 2000): 1971–82. http://dx.doi.org/10.1101/gad.14.15.1971.
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The initial steps of double-stranded break (DSB) repair by homologous recombination mediated by the 5′–3′ exonuclease/annealing protein pairs, RecE/RecT and Redα/Redβ, were analyzed. Recombination was RecA-independent and required the expression of both components of an orthologous pair, even when the need for exonuclease activity was removed by use of preresected substrates. The required orthologous function correlated with a specific protein–protein interaction, and recombination was favored by overexpression of the annealing protein with respect to the exonuclease. The need for both components of an orthologous pair was observed regardless of whether recombination proceeded via a single-strand annealing or a putative strand invasion mechanism. The DSB repair reactions studied here are reminiscent of the RecBCD/RecA reaction and suggest a general mechanism that is likely to be relevant to other systems, including RAD52 mediated recombination.
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Akyüz, Nuray, Gisa S. Boehden, Silke Süsse, Andreas Rimek, Ute Preuss, Karl-Heinz Scheidtmann, and Lisa Wiesmüller. "DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair." Molecular and Cellular Biology 22, no. 17 (September 1, 2002): 6306–17. http://dx.doi.org/10.1128/mcb.22.17.6306-6317.2002.
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ABSTRACT DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endonucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep process of tumor progression. For p53 direct regulatory roles in homologous recombination (HR) and in non-homologous end joining (NHEJ) were postulated. To systematically analyze the involvement of p53 in DSB repair, we generated a fluorescence-based assay system with a series of episomal and chromosomally integrated substrates for I-SceI meganuclease-triggered repair. Our data indicate that human wild-type p53, produced either stably or transiently in a p53-negative background, inhibits HR between substrates for conservative HR (cHR) and for gene deletions. NHEJ via microhomologies flanking the I-SceI cleavage site was also downregulated after p53 expression. Interestingly, the p53-dependent downregulation of homology-directed repair was maximal during cHR between sequences with short homologies. Inhibition was minimal during recombination between substrates that support reporter gene reconstitution by HR and NHEJ. p53 with a hotspot mutation at codon 281, 273, 248, 175, or 143 was severely defective in regulating DSB repair (frequencies elevated up to 26-fold). For the transcriptional transactivation-inactive variant p53(138V) a defect became apparent with short homologies only. These results suggest that p53 plays a role in restraining DNA exchange between imperfectly homologous sequences and thereby in suppressing tumorigenic genome rearrangements.
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Clikeman, Jennifer A., Sarah L. Wheeler, and Jac A. Nickoloff. "Efficient Incorporation of Large (>2 kb) Heterologies Into Heteroduplex DNA: Pms1/Msh2-Dependent and -Independent Large Loop Mismatch Repair in Saccharomyces cerevisiae." Genetics 157, no. 4 (April 1, 2001): 1481–91. http://dx.doi.org/10.1093/genetics/157.4.1481.
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Abstract DNA double-strand break (DSB) repair in yeast is effected primarily by gene conversion. Conversion can conceivably result from gap repair or from mismatch repair of heteroduplex DNA (hDNA) in recombination intermediates. Mismatch repair is normally very efficient, but unrepaired mismatches segregate in the next cell division, producing sectored colonies. Conversion of small heterologies (single-base differences or insertions <15 bp) in meiosis and mitosis involves mismatch repair of hDNA. The repair of larger loop mismatches in plasmid substrates or arising by replication slippage is inefficient and/or independent of Pms1p/Msh2p-dependent mismatch repair. However, large insertions convert readily (without sectoring) during meiotic recombination, raising the question of whether large insertions convert by repair of large loop mismatches or by gap repair. We show that insertions of 2.2 and 2.6 kbp convert efficiently during DSB-induced mitotic recombination, primarily by Msh2p- and Pms1p-dependent repair of large loop mismatches. These results support models in which Rad51p readily incorporates large heterologies into hDNA. We also show that large heterologies convert more frequently than small heterologies located the same distance from an initiating DSB and propose that this reflects Msh2-independent large loop-specific mismatch repair biased toward loop loss.
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Torrecilla, Ignacio, Judith Oehler, and Kristijan Ramadan. "The role of ubiquitin-dependent segregase p97 (VCP or Cdc48) in chromatin dynamics after DNA double strand breaks." Philosophical Transactions of the Royal Society B: Biological Sciences 372, no. 1731 (August 28, 2017): 20160282. http://dx.doi.org/10.1098/rstb.2016.0282.
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DNA double strand breaks (DSBs) are the most cytotoxic DNA lesions and, if not repaired, lead to chromosomal rearrangement, genomic instability and cell death. Cells have evolved a complex network of DNA repair and signalling molecules which promptly detect and repair DSBs, commonly known as the DNA damage response (DDR). The DDR is orchestrated by various post-translational modifications such as phosphorylation, methylation, ubiquitination or SUMOylation. As DSBs are located in complex chromatin structures, the repair of DSBs is engineered at two levels: (i) at sites of broken DNA and (ii) at chromatin structures that surround DNA lesions. Thus, DNA repair and chromatin remodelling machineries must work together to efficiently repair DSBs. Here, we summarize the current knowledge of the ubiquitin-dependent molecular unfoldase/segregase p97 (VCP in vertebrates and Cdc48 in worms and lower eukaryotes) in DSB repair. We identify p97 as an essential factor that regulates DSB repair. p97-dependent extraction of ubiquitinated substrates mediates spatio-temporal protein turnover at and around the sites of DSBs, thus orchestrating chromatin remodelling and DSB repair. As p97 is a druggable target, p97 inhibition in the context of DDR has great potential for cancer therapy, as shown for other DDR components such as PARP, ATR and CHK1. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.
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Llorente, Bertrand, and Lorraine S. Symington. "The Mre11 Nuclease Is Not Required for 5′ to 3′ Resection at Multiple HO-Induced Double-Strand Breaks." Molecular and Cellular Biology 24, no. 21 (November 1, 2004): 9682–94. http://dx.doi.org/10.1128/mcb.24.21.9682-9694.2004.
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ABSTRACT Current hypotheses suggest the Mre11 nuclease activity could be directly involved in double-strand break (DSB) resection in the presence of a large number of DSBs or limited to processing abnormal DNA ends. To distinguish between these possibilities, we used two methods to create large numbers of DSBs in Saccharomyces cerevisiae chromosomes, without introducing other substrates for the Mre11 nuclease. Multiple DSBs were created either by expressing the HO endonuclease in strains containing several HO cut sites embedded within randomly dispersed Ty1 elements or by phleomycin treatment. Analysis of resection by single-strand DNA formation in these systems showed no difference between strains containing MRE11 or the mre11-D56N nuclease defective allele, suggesting that the Mre11 nuclease is not involved in the extensive 5′ to 3′ resection of DSBs. We postulate that the ionizing radiation (IR) sensitivity of mre11 nuclease-defective mutants results from the accumulation of IR-induced DNA damage that is normally processed by the Mre11 nuclease. We also report that the processivity of 5′ to 3′ DSB resection and the yield of repaired products are affected by the number of DSBs in a dose-dependent manner. Finally, we show that the exonuclease Exo1 is involved in the processivity of 5′ to 3′ resection of an HO-induced DSB at the MAT locus.
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Itokawa, Hiroshi, Akiko Nomachi, Nobuaki Yasutake, Tatsuya Ishida, Takashi Fukushima, Hideaki Harakawa, Yoshimasa Kawase, Atsushi Azuma, and Ichiro Mizushima. "Pattern Dependence of Epitaxial-Realignment in Direct Silicon Bonded (DSB) Substrates with Hybrid Crystal Orientation." ECS Transactions 13, no. 1 (December 18, 2019): 321–28. http://dx.doi.org/10.1149/1.2911513.
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Meyer, Damon, Becky Xu Hua Fu, and Wolf-Dietrich Heyer. "DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae." Proceedings of the National Academy of Sciences 112, no. 50 (November 25, 2015): E6907—E6916. http://dx.doi.org/10.1073/pnas.1507833112.
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Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.
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Mosbech, Anna, Claudia Lukas, Simon Bekker-Jensen, and Niels Mailand. "The Deubiquitylating Enzyme USP44 Counteracts the DNA Double-strand Break Response Mediated by the RNF8 and RNF168 Ubiquitin Ligases." Journal of Biological Chemistry 288, no. 23 (April 24, 2013): 16579–87. http://dx.doi.org/10.1074/jbc.m113.459917.
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Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29 displayed nonspecific reactivity toward ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting considerable functional redundancy among cellular DUBs that restrict ubiquitin-dependent protein assembly at DSBs. Our findings implicate USP44 in negative regulation of the RNF8/RNF168 pathway and illustrate the usefulness of DUB overexpression screens for identification of antagonizers of ubiquitin-dependent cellular responses.
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Campbell, Amy E., Catarina Ferraz Franco, Ling-I. Su, Emma K. Corbin, Simon Perkins, Anton Kalyuzhnyy, Andrew R. Jones, Philip J. Brownridge, Neil D. Perkins, and Claire E. Eyers. "Temporal modulation of the NF-κB RelA network in response to different types of DNA damage." Biochemical Journal 478, no. 3 (February 10, 2021): 533–51. http://dx.doi.org/10.1042/bcj20200627.
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Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA.
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Choi, Jae-Yeon, Raymond Black, HeeJung Lee, James Di Giovanni, Robert C. Murphy, Choukri Ben Mamoun, and Dennis R. Voelker. "An improved and highly selective fluorescence assay for measuring phosphatidylserine decarboxylase activity." Journal of Biological Chemistry 295, no. 27 (May 19, 2020): 9211–22. http://dx.doi.org/10.1074/jbc.ra120.013421.
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Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/β-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.
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Jessulat, Matthew, Ramy H. Malty, Diem-Hang Nguyen-Tran, Viktor Deineko, Hiroyuki Aoki, James Vlasblom, Katayoun Omidi, et al. "Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining." Molecular and Cellular Biology 35, no. 14 (May 11, 2015): 2448–63. http://dx.doi.org/10.1128/mcb.00007-15.
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The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.
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Tadi, Satish Kumar, Robin Sebastian, Sumedha Dahal, Ravi K. Babu, Bibha Choudhary, and Sathees C. Raghavan. "Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions." Molecular Biology of the Cell 27, no. 2 (January 15, 2016): 223–35. http://dx.doi.org/10.1091/mbc.e15-05-0260.
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Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.
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McMurry, M. T., C. Hernandez-Munain, P. Lauzurica, and M. S. Krangel. "Enhancer control of local accessibility to V(D)J recombinase." Molecular and Cellular Biology 17, no. 8 (August 1997): 4553–61. http://dx.doi.org/10.1128/mcb.17.8.4553.
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We have studied the role of transcriptional enhancers in providing recombination signal sequence (RSS) accessibility to V(D)J recombinase by examining mice carrying a transgenic human T-cell receptor (TCR) delta gene minilocus. This transgene is composed of unrearranged variable (Vdelta and Vdelta2), diversity (Ddelta3), joining (Jdelta1 and Jdelta3), and constant (Cdelta) gene segments. Previous data indicated that with the TCR delta enhancer (Edelta) present in the Jdelta3-Cdelta intron, V(D)J recombination proceeds stepwise, first V to D and then VD to J. With the enhancer deleted or mutated, V-to-D rearrangement is intact, but VD-to-J rearrangement is inhibited. We proposed that Edelta is necessary for J segment but not D segment accessibility and that J segment inaccessibility in the enhancerless minilocus resulted in the observed V(D)J recombination phenotype. In this study, we tested this notion by using ligation-mediated PCR to assess the formation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs 3' of Ddelta3 and 5' of Jdelta1. In five lines of mice carrying multicopy integrants of constructs that either lacked Edelta or carried an inactivated Edelta, the frequency of DSBs 5' of Jdelta1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3' of Ddelta3 was unaffected. We interpret these results to indicate that Edelta is required for Jdelta1 but not Ddelta3 accessibility within the minilocus, and we conclude that enhancers regulate V(D)J recombination by providing local accessibility to the recombinase. cis-acting elements other than Edelta must maintain Ddelta3 in an accessible state in the absence of Edelta. The analysis of DSB formation in a single-copy minilocus integrant indicates that efficient DSB formation at the accessible RSS 3' of Ddelta3 requires an accessible partner RSS, arguing that RSS synapsis is required for DSB formation in chromosomal substrates in vivo.
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Stephanou, Nicolas C., Feng Gao, Paola Bongiorno, Sabine Ehrt, Dirk Schnappinger, Stewart Shuman, and Michael S. Glickman. "Mycobacterial Nonhomologous End Joining Mediates Mutagenic Repair of Chromosomal Double-Strand DNA Breaks." Journal of Bacteriology 189, no. 14 (May 11, 2007): 5237–46. http://dx.doi.org/10.1128/jb.00332-07.
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ABSTRACT Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis ΔrecA Δku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.
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Miller, Elizabeth M., Heather L. Hough, Jennifer W. Cho, and Jac A. Nickoloff. "Mismatch Repair by Efficient Nick-Directed, and Less Efficient Mismatch-Specific, Mechanisms in Homologous Recombination Intermediates in Chinese Hamster Ovary Cells." Genetics 147, no. 2 (October 1, 1997): 743–53. http://dx.doi.org/10.1093/genetics/147.2.743.
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Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in ∼10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A → G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was >80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks.
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Karakaidos, Panagiotis, Christina Kryou, Nikiana Simigdala, Apostolos Klinakis, and Ioanna Zergioti. "Laser Bioprinting of Cells Using UV and Visible Wavelengths: A Comparative DNA Damage Study." Bioengineering 9, no. 8 (August 9, 2022): 378. http://dx.doi.org/10.3390/bioengineering9080378.
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Laser-based techniques for printing cells onto different substrates with high precision and resolution present unique opportunities for contributing to a wide range of biomedical applications, including tissue engineering. In this study, laser-induced forward transfer (LIFT) printing was employed to rapidly and accurately deposit patterns of cancer cells in a non-contact manner, using two different wavelengths, 532 and 355 nm. To evaluate the effect of LIFT on the printed cells, their growth and DNA damage profiles were assessed and evaluated quantitatively over several days. The damaging effect of LIFT-printing was thoroughly investigated, for the first time at a single cell level, by counting individual double strand breaks (DSB). Overall, we found that LIFT was able to safely print patterns of breast cancer cells with high viability with little or no heat or shear damage to the cells, as indicated by unperturbed growth and negligible gross DNA damage.
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Li, Fang. "Abstract 6214: FANCA promotes transcription-coupled homologous recombination by catalyzing R-loops formation." Cancer Research 83, no. 7_Supplement (April 4, 2023): 6214. http://dx.doi.org/10.1158/1538-7445.am2023-6214.
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Abstract R loops arise from hybridization of RNA transcripts with template DNA during transcription, which are not only cause DNA damage in certain contexts as cancer hallmarks but also be important regulators of cellular processes. Recent findings revealed that DNA double-strand breaks (DSBs) elicit RNA-DNA hybrid formation. The RNA moiety of the R-loop displays an extraordinary potential to regulate multiple steps of homologous recombination (HR) by substituting for the double-stranded DNA (dsDNA) substrate on which DSB repair processes naturally occur. Here, we use the Damage At RNA Transcription assay to reveal colocalization of FANCA with R-loops in a highly transcribed genomic locus upon DNA damage. We further demonstrate FANCA participates in forming R-loops in the initial DSBs repair stage and assists in removing R-loops later. Besides, we find that highly purified human FANCA anneals synthetic single-stranded RNA (ssRNA) and ssDNA species to R-loops and binds R-loop substrates with high affinity, preferring guanine-rich sequences in vitro. Importantly, we illustrate that FANCA promotes HR efficiency via catalyzation of DNA: RNA hybrids at DSBs sites. Finally, a series of RNA and R-loop substrates are found to stimulate ID2 monoubiquitination, with activity corresponding to remove transient R- loops in later HR stage. In summary, our results support a mechanism whereby FANCA promotes the formation of temporary R-loops by annealing ssRNA and ssDNA species at DSBs in transcribed regions, thereby stimulating HR. Citation Format: Fang Li. FANCA promotes transcription-coupled homologous recombination by catalyzing R-loops formation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6214.
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Degorre, Charlotte M., Steven Lohard, and Philip J. Tofilon. "Abstract 2406: Targeting prmt5 inhibits DNA repair and enhances the radiosensitivity of GBM cells." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2406. http://dx.doi.org/10.1158/1538-7445.am2023-2406.
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Abstract Glioblastoma (GBM) is considered highly radioresistant. To improve the efficacy of GBM radiotherapy, one approach is the addition of a targeted radiosensitizer, which requires an understanding of the molecules that determine GBM radioresponse. Along these lines, PRMT5 is a type II arginine methyltransferase that symmetrically dimethylates histone and non-histone substrates to regulate chromatin structure and mRNA splicing, two parameters that can influence radiosensitivity. As an initial test of PRMT5 as a target for GBM radiosensitization, PRMT5 knockdown using siRNA was shown to increase the in vitro radiosensitivity of U251 GBM cells. PRMT5 was then targeted using the small molecule inhibitor LLY-283, which penetrates the blood brain barrier. Exposure of U251 cells to LLY-283 (24h) reduced the levels of symmetric dimethylated arginine as determined by western blot, indicating an inhibition of PRMT5 activity. Based on clonogenic analysis, LLY-283 exposure beginning 1h prior to irradiation significantly enhanced the radiosensitivity of U251 cells. Whereas treatment of the normal fibroblast cell line MRC9 with LLY-283 reduced PRMT5 activity, no effect was detected on radiosensitivity, suggesting tumor cell selective radiosensitization. As an initial investigation of the radiosensitizing mechanism, U251 were treated with LLY-283 1h prior irradiation (2Gy) and the number of γH2AX foci, marker for DNA double strand breaks (DSBs), was determined. Whereas LLY-283 had no effect on the initial level of radiation-induced γH2AX foci, the dispersal of foci was significantly delayed in LLY-283 treated cells. These results suggest that LLY-283 inhibits the repair of radiation-induced DSBs. The neutral comet assay, an alternative measure of radiation-induced DSBs showed that LLY-283 delivered 1h prior irradiation (10Gy) significantly increased the comet tail moment detected at 6 and 24h as compared to 10Gy only, consistent with an inhibition of DSB repair. Based on these two approaches LLY-283-induced radiosensitization appears to be mediated by an inhibition of DSB repair. Extending these studies to GBM stem-like cell lines (GSCs) showed that LLY-283 inhibited PRMT5 activity and when delivered 1h prior to irradiation enhanced radiosensitivity. Analysis of γH2AX foci showed a delay in foci dispersal in LLY-283 treated cells consistent with an inhibition of DSB repair. Because a 1h LLY-283 pretreatment was sufficient for the inhibition of DSB repair and enhanced radiosensitivity, it appears that the radiosensitization was primarily the result of altered chromatin function rather than changes in gene expression due to alternative mRNA splicing. Altogether, these results suggest targeting PRMT5 as a potential strategy for enhancing the radiosensitivity of GBMs. Citation Format: Charlotte M. Degorre, Steven Lohard, Philip J. Tofilon. Targeting prmt5 inhibits DNA repair and enhances the radiosensitivity of GBM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2406.
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Prado, F., and A. Aguilera. "Role of reciprocal exchange, one-ended invasion crossover and single-strand annealing on inverted and direct repeat recombination in yeast: different requirements for the RAD1, RAD10, and RAD52 genes." Genetics 139, no. 1 (January 1, 1995): 109–23. http://dx.doi.org/10.1093/genetics/139.1.109.
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Abstract We have constructed novel DNA substrates (one inverted and three direct repeats) based on the same 0.6-kb repeat sequence to study deletions and inversions in Saccharomyces cerevisiae. Spontaneous deletions occur six to eight times more frequently than inversions, irrespective of the distance between the repeats. This difference can be explained by the observation that deletion events can be mediated by a recombination mechanism that can initiate within the intervening sequence of the repeats. Spontaneous and double-strand break (DSB)-induced deletions occur as RAD52-dependent and RAD52-independent events. Those deletion events initiated through a DSB in the unique intervening sequence require the Rad1/Rad10 endonuclease only if the break is distantly located from the flanking DNA repeats. We propose that deletions can occur as three types of recombination events: the conservative RAD52-dependent reciprocal exchange and the nonconservative events, one-ended invasion crossover, and single-strand annealing (SSA). We suggest that one-ended invasion is RAD52 dependent, whereas SSA is RAD52 independent. Whereas deletions, like inversions, occur through reciprocal exchange, deletions can also occur through SSA or one-ended invasion. We propose that the contribution of reciprocal exchange and one-ended invasion crossover vs. SSA events to overall spontaneous deletions is a feature specific for each repeat system, determined by the initiation event and the availability of the Rad52 protein. We discuss the role of the Rad1/Rad10 endonuclease on the initial steps of one-ended invasion crossover and SSA as a function of the location of the initiation event relative to the repeats. We also show that the frequency of recombination between repeats is the same independent of their location (whether on circular plasmids, linear minichromosomes, or natural chromosomes) and have similar RAD52 dependence.
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Toma, Aya, Tomio Takahashi, Yusuke Sato, Sakurako Goto-Ito, Atsushi Yamagata, and Shuya Fukai. "Ubiquitin recognition by UBZ and UMI domains for DNA damage response." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1642. http://dx.doi.org/10.1107/s2053273314083570.
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Double-strand break (DSB) and interstrand crosslink (ICL) are serious damages in DNA. Responses to these DNA damages include ubiquitination of damaged chromatin and other substrates, which recruit protein complexes required for DNA repair. Therefore, many proteins involved in DNA damage response contain ubiquitin-binding modules. For instance, a ubiquitin ligase RNF168, which catalyzes K63-linked polyubiquitination of histone H2A, contains two types of ubiquitin binding motifs, MIU (motif interacting with ubiquitin) and UIM (UIM and MIU-related Ub-binding domain). FAAP20, which recruits Fanconi anemia proteins (crosslink-repair factors), contains a UBZ (ubiquitin-binding zinc finger) domain. To date, mechanisms for ubiquitin recognition by UMI and UBZ domains have remained unclear. In this study, we determined crystal structures of RNF168 UMI and FAAP20 UBZ in complex with ubiquitin at 1.9 Å resolutions, respectively. SPR analyses using UMI and UBZ mutants, which were designed to disrupt Ub binding, confirmed that the observed interactions between Ub and UMI or UBZ are critical for binding. Our structure and the accompanying in-vitro structure-based mutagenesis experiments reveal the structural basis of these important recognition events.
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Choi, Jihyun, Muwen Kong, Danielle N. Gallagher, Kevin Li, Gabriel Bronk, Yiting Cao, Eric Greene, and James E. Haber. "Repair of mismatched templates during Rad51-dependent Break-Induced Replication." PLOS Genetics 18, no. 9 (September 2, 2022): e1010056. http://dx.doi.org/10.1371/journal.pgen.1010056.
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Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3’ end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. BIR still occurs about 10% as often when every 6th base is mismatched as with a perfectly matched donor. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. A donor with all 10 mismatches clustered at the 3’ invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5’ end. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but mismatch position-specific effects are also important. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6th base was mismatched. Now, using substrates of either 90 or 108 nt–the latter being the size of the in vivo templates–we find that in vitro D-loop results are very similar to the in vivo results. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3’ end. Mismatch incorporation depends on the 3’→ 5’ proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Surprisingly, the probability of a mismatch 27 nt from the 3’ end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ “chews back” the 3’ end of the invading strand without any mismatch-dependent cues from the strand invasion structure. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3’ end of the donor.
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Yu, Tai-Yuan, Michael T. Kimble, and Lorraine S. Symington. "Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection." Proceedings of the National Academy of Sciences 115, no. 51 (December 3, 2018): E11961—E11969. http://dx.doi.org/10.1073/pnas.1816539115.
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The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.
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Pan, Hai, Miao Jin, Ashwin Ghadiyaram, Parminder Kaur, Henry E. Miller, Hai Minh Ta, Ming Liu, et al. "Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA." Nucleic Acids Research 48, no. 10 (April 30, 2020): 5639–55. http://dx.doi.org/10.1093/nar/gkaa284.
Full textAbstract:
Abstract Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.
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Zhang, Ming, and Patrick C. Swanson. "Recognition and cleavage of cryptic recombination signal sequences identified from lymphoid malignancies (49.20)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S86—S87. http://dx.doi.org/10.4049/jimmunol.178.supp.49.20.
Full textAbstract:
Abstract Immune repertoire diversity is largely achieved by V (D) J recombination, during which antigen receptor genes are assembled from arrays of component gene segments by site-specific DNA rearrangement. This process is initiated when the RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) abutting antigen receptor coding segments. However, the RAG proteins occasionally mistarget DNA sequences outside of antigen receptor loci and promote chromosomal translocations and deletions, which are among the major events that initiate neoplasia in lymphoid organs. Previous studies have identified various RSS-like sequences involved in chromosomal translocations, including LMO2, TAL1, Ttg-1, and Hox11, and the interstitial deletion at 1p32 involving SIL/SCL. Here we have used electrophoretic mobility shift assays and in vitro cleavage assays to study RAG-mediated binding and cleavage to the oligonucleotide substrates containing these cryptic RSSs. We find that RAG proteins can cleave LMO2, TAL1, Ttg-1, and SIL in vitro, and introduce DSBs at these sites which can be detected by ligation-mediated PCR in cell culture. In contrast, Hox11 and SCL are nicked but fail to support DSB formation both in vitro and in cell culture. These data raise the possibility that Hox11 and SCL may be cleaved by an as-yet unidentified mechanism. This research is supported by American Cancer Society grant RSG-01-020-05-LIB to P.C.S.
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Pastwa, Elzbieta, Tomasz Poplawski, Agnieszka Czechowska, Mariusz Malinowski, and Janusz Blasiak. "Non-homologous DNA End Joining Repair in Normal and Leukemic Cells Depends on the Substrate Ends." Zeitschrift für Naturforschung C 60, no. 5-6 (June 1, 2005): 493–500. http://dx.doi.org/10.1515/znc-2005-5-619.
Full textAbstract:
Double-strand breaks (DSBs) are the most serious DNA damage which, if unrepaired or misrepaired, may lead to cell death, genomic instability or cancer transformation. In human cells they can be repaired mainly by non-homologous DNA end joining (NHEJ). The efficacy of NHEJ pathway was examined in normal human lymphocytes and K562 myeloid leukemic cells expressing the BCR/ABL oncogenic tyrosine kinase activity and lacking p53 tumor suppressor protein. In our studies we employed a simple and rapid in vitro DSB end joining assay based on fluorescent detection of repair products. Normal and cancer cells were able to repair DNA damage caused by restriction endonucleases, but the efficiency of the end joining was dependent on the type of cells and the structure of DNA ends. K562 cells displayed decreased NHEJ activity in comparison to normal cells for 5′ complementary DNA overhang. For blunt-ended DNA there was no significant difference in end joining activity. Both kinds of cells were found about 10-fold more efficient for joining DNA substrates with compatible 5′ overhangs than those with blunt ends. Our recent findings have shown that stimulation of DNA repair could be involved in the drug resistance of BCR/ABL-positive cells in anticancer therapy. For the first time the role of STI571 was investigated, a specific inhibitor of BCR/ABL oncogenic protein approved for leukemia treatment in the NHEJ pathway. Surprisingly, STI571 did not change the response of BCR/ABL-positive K562 cells in terms of NHEJ for both complementary and blunt ends. Our results suggest that the various responses of the cells to DNA damage via NHEJ can be correlated with the differences in the genetic constitution of human normal and cancer cells. However, the role of NHEJ in anticancer drug resistance in BCR/ABL-positive cells is questionable.
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Gamble, Dionna, Samantha Shaltz, and Sue Jinks-Robertson. "Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast." G3: Genes|Genomes|Genetics 10, no. 10 (August 21, 2020): 3821–29. http://dx.doi.org/10.1534/g3.120.401603.
Full textAbstract:
Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae. Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3′ overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3′ end affects recombination outcomes and whether the presence of a 3′ vs. 5′ overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers vs. noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3′ end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3′ vs. 5′ overhangs differs, we compared the well-studied 3′ overhang created by I-SceI to a 5′ overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.
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Segal, D. J., A. F. Faruqi, P. M. Glazer, and D. Carroll. "Processing of targeted psoralen cross-links in Xenopus oocytes." Molecular and Cellular Biology 17, no. 11 (November 1997): 6645–52. http://dx.doi.org/10.1128/mcb.17.11.6645.
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Psoralen cross-links have been shown to be both mutagenic and recombinagenic in bacterial, yeast, and mammalian cells. Double-strand breaks (DSBs) have been implicated as intermediates in the removal of psoralen cross-links. Recent work has suggested that site-specific mutagenesis and recombination might be achieved through the use of targeted psoralen adducts. The fate of plasmids containing psoralen adducts was evaluated in Xenopus oocytes, an experimental system that has well-characterized recombination capabilities and advantages in the analysis of intermediates in DNA metabolism. Psoralen adducts were delivered to a specific site by a triplex-forming oligonucleotide. These lesions are clearly recognized and processed in oocytes, since mutagenesis was observed at the target site. The spectrum of induced mutations was compared with that found in similar studies in mammalian cells. Plasmids carrying multiple random adducts were preferentially degraded, perhaps due to the introduction of DSBs. However, when DNAs carrying site-specific adducts were examined, no plasmid loss was observed and removal of cross-links was found to be very slow. Sensitive assays for DSB-dependent homologous recombination were performed with substrates with one or two cross-link sites. No adduct-stimulated recombination was observed with a single lesion, and only very low levels were observed with paired lesions, even when a large proportion of the cross-links was removed by the oocytes. We conclude that DSBs or other recombinagenic structures are not efficiently formed at psoralen adducts in Xenopus oocytes. While psoralen is not a promising reagent for stimulating site-specific recombination, it is effective in inducing targeted mutations.
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Raczko, Anna M., Janusz M. Bujnicki, Marcin Pawłowski, Renata Godlewska, Magdalena Lewandowska, and Elżbieta K. Jagusztyn-Krynicka. "Characterization of new DsbB-like thiol-oxidoreductases of Campylobacter jejuni and Helicobacter pylori and classification of the DsbB family based on phylogenomic, structural and functional criteria." Microbiology 151, no. 1 (January 1, 2005): 219–31. http://dx.doi.org/10.1099/mic.0.27483-0.
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In Gram-negative bacterial cells, disulfide bond formation occurs in the oxidative environment of the periplasm and is catalysed by Dsb (disulfide bond) proteins found in the periplasm and in the inner membrane. In this report the identification of a new subfamily of disulfide oxidoreductases encoded by a gene denoted dsbI, and functional characterization of DsbI proteins from Campylobacter jejuni and Helicobacter pylori, as well as DsbB from C. jejuni, are described. The N-terminal domain of DsbI is related to DsbB proteins and comprises five predicted transmembrane segments, while the C-terminal domain is predicted to locate to the periplasm and to fold into a β-propeller structure. The dsbI gene is co-transcribed with a small ORF designated dba ( dsbI-accessory). Based on a series of deletion and complementation experiments it is proposed that DsbB can complement the lack of DsbI but not the converse. In the presence of DsbB, the activity of DsbI was undetectable, hence it probably acts only on a subset of possible substrates of DsbB. To reconstruct the principal events in the evolution of DsbB and DsbI proteins, sequences of all their homologues identifiable in databases were analysed. In the course of this study, previously undetected variations on the common thiol-oxidoreductase theme were identified, such as development of an additional transmembrane helix and loss or migration of the second pair of Cys residues between two distinct periplasmic loops. In conjunction with the experimental characterization of two members of the DsbI lineage, this analysis has resulted in the first comprehensive classification of the DsbB/DsbI family based on structural, functional and evolutionary criteria.
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Séguéla-Arnaud, Mathilde, Wayne Crismani, Cécile Larchevêque, Julien Mazel, Nicole Froger, Sandrine Choinard, Afef Lemhemdi, et al. "Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM." Proceedings of the National Academy of Sciences 112, no. 15 (March 30, 2015): 4713–18. http://dx.doi.org/10.1073/pnas.1423107112.
Full textAbstract:
Meiotic crossovers (COs) have two important roles, shuffling genetic information and ensuring proper chromosome segregation. Despite their importance and a large excess of precursors (i.e., DNA double-strand breaks, DSBs), the number of COs is tightly regulated, typically one to three per chromosome pair. The mechanisms ensuring that most DSBs are repaired as non-COs and the evolutionary forces imposing this constraint are poorly understood. Here we identified Topoisomerase3α (TOP3α) and the RECQ4 helicases—the Arabidopsis slow growth suppressor 1 (Sgs1)/Bloom syndrome protein (BLM) homologs—as major barriers to meiotic CO formation. First, the characterization of a specific TOP3α mutant allele revealed that, in addition to its role in DNA repair, this topoisomerase antagonizes CO formation. Further, we found that RECQ4A and RECQ4B constitute the strongest meiotic anti-CO activity identified to date, their concomitant depletion leading to a sixfold increase in CO frequency. In both top3α and recq4ab mutants, DSB number is unaffected, and extra COs arise from a normally minor pathway. Finally, both TOP3α and RECQ4A/B act independently of the previously identified anti-CO Fanconi anemia of complementation group M (FANCM) helicase. This finding shows that several parallel pathways actively limit CO formation and suggests that the RECQA/B and FANCM helicases prevent COs by processing different substrates. Despite a ninefold increase in CO frequency, chromosome segregation was unaffected. This finding supports the idea that CO number is restricted not because of mechanical constraints but likely because of the long-term costs of recombination. Furthermore, this work demonstrates how manipulating a few genes holds great promise for increasing recombination frequency in plant-breeding programs.
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Altland, James E., M. Gabriela Buamscha, and Donald A. Horneck. "Substrate pH Affects Nutrient Availability in Fertilized Douglas Fir Bark Substrates." HortScience 43, no. 7 (December 2008): 2171–78. http://dx.doi.org/10.21273/hortsci.43.7.2171.
Full textAbstract:
An experiment was conducted to determine how pH and nutrient availability in douglas fir bark (DFB) substrates respond to lime and sulfur (S) rates. The treatment design was a two-by-nine factorial arrangement with two substrate types and nine pH-altering amendments. The two substrates were 100% DFB or 75 DFB:15 sphagnum peatmoss:10 pumice (by volume). Substrate pH-altering amendments included elemental S amended at either 0.6 or 2.4 kg·m−3; calcium carbonate amended at 0.6, 1.5, and 5.9 kg·m−3; calcium hydroxide amended at 4.4, 8.9, or 23.7 kg·m−3; and a nonamended control. All substrates were amended by incorporating 0.9 kg·m−3 Micromax micronutrients before potting and topdressing 8 g/pot of 14N–4.2P–11.6K Osmocote controlled-release fertilizer after potting. A group of controls was also maintained for each substrate that received no fertilizer amendment (no S, lime, Micromax, or Osmocote). Four containers of each treatment were randomly selected and harvested 4 and 8 weeks after potting. Amendment with S decreased pH with increasing rate, whereas both lime types increased pH with increasing rate. The two substrates in general responded similarly to S and lime amendments, although there were some significant effects and interactions caused by substrate type. Ammonium-N and NO3-N both decreased exponentially with increasing substrate pH, whereas water-extractable phosphorus decreased linearly with increasing pH. Water-extractable potassium, calcium, magnesium, and sodium responded quadratically to increasing pH by initially decreasing and then increasing. The micronutrients boron and iron decreased with increasing pH, whereas DTPA extractions of manganese, zinc, and copper initially increased and then decreased over the range of observed pH.
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Roy, Subir, S. Rangaswamy Reddy, P. Sindhuja, Dipak Das, and V. V. Bhauprasad. "AlPO4-C Composite Coating on Ni-based Super Alloy Substrates for High Emissivity Applications : Experimentation on Dip Coating and Spray Coating." Defence Science Journal 66, no. 4 (June 28, 2016): 425. http://dx.doi.org/10.14429/dsj.66.10220.
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<p>High emissivity coating was developed on Ni-based super alloy substrates by dip coating and spray coating technique using a chemical precursor sol. The coating material was characterised thoroughly by XRD, SEM, TEM and XPS analyses. Characterisation results showed the presence of nano carbon in the AlPO4 matrix which imparted high emissivity to the coating. Emissivity of the coating varied from 0.6 to 0.9 in the wave length range : 2 µm - 25 µm depending on the thickness of the multilayered coating. Spray coating was very effective for coating the bigger substrates and TPS panels. Emissivity offered by the spray coated substrates was little lower compared to the dip coated substrates. Emissivity offered by the spray coated substrates was little lower compared to the dip coated substrates. Cyclic oxidation performances of the coated substrates at 800 °C and 1000 °C for 100 hrs of thermal exposure were recorded and compared with that of the bare substrate. The emissivity coating was found to offer substantial oxidation resistance to the base substrate at high temperatures.</p>
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Gabriel, Magdalena Zazirska, James E. Altland, and James S. Owen. "The Effect of Physical and Hydraulic Properties of Peatmoss and Pumice on Douglas Fir Bark Based Soilless Substrates." HortScience 44, no. 3 (June 2009): 874–78. http://dx.doi.org/10.21273/hortsci.44.3.874.
Full textAbstract:
Douglas fir [Pseudotsuga menziesii Mirb. (Franco)] bark (DFB), sphagnum peatmoss, and pumice are the most common substrate components used in the Oregon nursery industry. The objective of this study was to document the effect of peat and pumice addition on the physical and hydrological properties of DFB soilless substrates. A secondary objective was to determine if measured properties of mixed soilless substrates can be accurately predicted from the known properties of the individual components. Treatment design was a 3 × 3 factorial with three rates each of sphagnum peatmoss and pumice (0%, 15%, and 30% by vol.) added to DFB. The resulting nine substrates were measured for total porosity, air space, container capacity, and bulk density using porometers. Moisture characteristic curves were generated by measuring water content along a continuous column. Adding pumice to DFB decreased total porosity, container capacity, available water, and water-buffering capacity but increased bulk density. Adding peatmoss to DFB increased total porosity, container capacity, and available water but decreased air space and bulk density. Comparison of predicted values against measured values indicated that bulk density could be predicted reliably; however, all other physical properties could not be accurately predicted.
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Ndaru, Elias, Rachel-Ann A. Garibsingh, Laura Zielewicz, Avner Schlessinger, and Christof Grewer. "Interaction of the neutral amino acid transporter ASCT2 with basic amino acids." Biochemical Journal 477, no. 8 (April 27, 2020): 1443–57. http://dx.doi.org/10.1042/bcj20190859.
Full textAbstract:
Glutamine transport across cell membranes is performed by a variety of transporters, including the alanine serine cysteine transporter 2 (ASCT2). The substrate-binding site of ASCT2 was proposed to be specific for small amino acids with neutral side chains, excluding basic substrates such as lysine. A series of competitive inhibitors of ASCT2 with low µM affinity were developed previously, on the basis of the 2,4-diaminobutyric acid (DAB) scaffold with a potential positive charge in the side chain. Therefore, we tested whether basic amino acids with side chains shorter than lysine can interact with the ASCT2 binding site. Molecular docking of L-1,3-diaminopropionic acid (L-DAP) and L-DAB suggested that these compounds bind to ASCT2. Consistent with this prediction, L-DAP and L-DAB, but not ornithine, lysine or D-DAP, elicited currents when applied to ASCT2-expressing cells. The currents were carried by anions and showed the hallmark properties of ASCT2 currents induced by transported substrates. The L-DAP response could be eliminated by a competitive ASCT2 inhibitor, suggesting that binding occurs at the substrate binding site. The KM for L-DAP was weakly voltage dependent. Furthermore, the pH dependence of the L-DAP response showed that the compound can bind in several protonation states. Together, these results suggest that the ASCT2 binding site is able to recognize L-amino acids with short, basic side chains, such as the L-DAP derivative β-N-methylamino-l-Alanine (BMAA), a well-studied neurotoxin. Our results expand the substrate specificity of ASCT2 to include amino acid substrates with positively charged side chains.