Journal articles on the topic 'DNA Single-strand Repair Pathway'
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1
Pandya, Gagan A., In-Young Yang, Arthur P. Grollman, and Masaaki Moriya. "Escherichia coli Responses to a Single DNA Adduct." Journal of Bacteriology 182, no. 23 (December 1, 2000): 6598–604. http://dx.doi.org/10.1128/jb.182.23.6598-6604.2000.
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ABSTRACT To study the mechanisms by which Escherichia colimodulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E. coli pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1,N 6-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF,recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.
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Zhao, Lei, Chengyu Bao, Yuxuan Shang, Xinye He, Chiyuan Ma, Xiaohua Lei, Dong Mi, and Yeqing Sun. "The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks." BioMed Research International 2020 (August 25, 2020): 1–12. http://dx.doi.org/10.1155/2020/4834965.
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Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.
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Yan, Hong, Thomas Toczylowski, Jill McCane, Chinyi Chen, and Shuren Liao. "Replication protein A promotes 5′→3′ end processing during homology-dependent DNA double-strand break repair." Journal of Cell Biology 192, no. 2 (January 24, 2011): 251–61. http://dx.doi.org/10.1083/jcb.201005110.
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Replication protein A (RPA), the eukaryotic single-strand deoxyribonucleic acid (DNA [ss-DNA])–binding protein, is involved in DNA replication, nucleotide damage repair, mismatch repair, and DNA damage checkpoint response, but its function in DNA double-strand break (DSB) repair is poorly understood. We investigated the function of RPA in homology-dependent DSB repair using Xenopus laevis nucleoplasmic extracts as a model system. We found that RPA is required for single-strand annealing, one of the homology-dependent DSB repair pathways. Furthermore, RPA promotes the generation of 3′ single-strand tails (ss-tails) by stimulating both the Xenopus Werner syndrome protein (xWRN)–mediated unwinding of DNA ends and the subsequent Xenopus DNA2 (xDNA2)–mediated degradation of the 5′ ss-tail. Purified xWRN, xDNA2, and RPA are sufficient to carry out the 5′-strand resection of DNA that carries a 3′ ss-tail. These results provide strong biochemical evidence to link RPA to a specific DSB repair pathway and reveal a novel function of RPA in the generation of 3′ ss-DNA for homology-dependent DSB repair.
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Li, Jinbao, Huize Sun, Yulin Huang, Yali Wang, Yuyan Liu, and Xuefeng Chen. "Pathways and assays for DNA double-strand break repair by homologous recombination." Acta Biochimica et Biophysica Sinica 51, no. 9 (July 10, 2019): 879–89. http://dx.doi.org/10.1093/abbs/gmz076.
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AbstractDouble strand breaks (DSBs) are the most detrimental type of DNA damage that must be repaired to ensure genome integrity and cell survival. Unrepaired or improperly repaired DSBs can potentially cause tumorigenesis or cell death. DSBs are primarily repaired by non-homologous end joining or homologous recombination (HR). The HR pathway is initiated by processing of the 5′-end of DSBs to generate 3′-end single-strand DNA (ssDNA). Furthermore, the intermediate is channeled to one of the HR sub-pathways, including: (i) double Holliday junction (dHJ) pathway, (ii) synthesis-dependent strand annealing (SDSA), (iii) break-induced replication (BIR), and (iv) single-strand annealing (SSA). In the dHJ sub-pathway, the 3′-ssDNA coated with Rad51 recombinase performs homology search and strand invasion, forming a displacement loop (D-loop). Capture of the second end by the D-loop generates a dHJ intermediate that is subsequently dissolved by DNA helicase or resolved by nucleases, producing non-crossover or crossover products. In SDSA, the newly synthesized strand is displaced from the D-loop and anneals to the end on the other side of the DSBs, producing non-crossovers. In contrast, BIR repairs one-end DSBs by copying the sequence up to the end of the template chromosome, resulting in translocation or loss of heterozygosity. SSA takes place when resection reveals flanking homologous repeats that can anneal, leading to deletion of the intervening sequences. A variety of reporter assays have been developed to monitor distinct HR sub-pathways in both Saccharomyces cerevisiae and mammals. Here, we summarize the principles and representative assays for different HR sub-pathways with an emphasis on the studies in the budding yeast.
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Preston, Christine R., William Engels, and Carlos Flores. "Efficient Repair of DNA Breaks in Drosophila: Evidence for Single-Strand Annealing and Competition With Other Repair Pathways." Genetics 161, no. 2 (June 1, 2002): 711–20. http://dx.doi.org/10.1093/genetics/161.2.711.
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Abstract We show evidence that DNA double-strand breaks induced in the Drosophila germ line can be repaired very efficiently by the single-strand annealing (SSA) mechanism. A double-strand break was made between two copies of a 1290-bp direct repeat by mobilizing a P transposon. In >80% of the progeny that acquired this chromosome, repair resulted in loss of the P element and loss of one copy of the repeat, as observed in SSA. The frequency of this repair was much greater than seen for gene conversion using an allelic template, which is only ∼7%. A similar structure, but with a smaller duplication of only 158 bp, also yielded SSA-like repair events, but at a reduced frequency, and gave rise to some products by repair pathways other than SSA. The 1290-bp repeats carried two sequence polymorphisms that were examined in the products. The allele nearest to a nick in the putative heteroduplex intermediate was lost most often. This bias is predicted by the SSA model, although other models could account for it. We conclude that SSA is the preferred repair pathway in Drosophila for DNA breaks between sequence repeats, and it competes with gene conversion by the synthesis-dependent strand annealing (SDSA) pathway.
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Lin, Yunfeng, Jude Raj, Jia Li, Anh Ha, Md Akram Hossain, Christine Richardson, Pinku Mukherjee, and Shan Yan. "APE1 senses DNA single-strand breaks for repair and signaling." Nucleic Acids Research 48, no. 4 (December 12, 2019): 1925–40. http://dx.doi.org/10.1093/nar/gkz1175.
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Abstract DNA single-strand breaks (SSBs) represent the most abundant type of DNA damage. Unrepaired SSBs impair DNA replication and transcription, leading to cancer and neurodegenerative disorders. Although PARP1 and XRCC1 are implicated in the SSB repair pathway, it remains unclear how SSB repair and SSB signaling pathways are coordinated and regulated. Using Xenopus egg extract and in vitro reconstitution systems, here we show that SSBs are first sensed by APE1 to initiate 3′–5′ SSB end resection, followed by APE2 recruitment to continue SSB end resection. Notably, APE1’s exonuclease activity is critical for SSB repair and SSB signaling pathways. An APE1 exonuclease-deficient mutant identified in somatic tissue from a cancer patient highlighted the significance of APE1 exonuclease activity in cancer etiology. In addition, APE1 interacts with APE2 and PCNA, although PCNA is dispensable for APE1’s exonuclease activity. Taken together, we propose a two-step APE1/APE2-mediated mechanism for SSB end resection that couples DNA damage response with SSB repair in a eukaryotic system.
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Davis, Luther, and Nancy Maizels. "Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair." Proceedings of the National Academy of Sciences 111, no. 10 (February 20, 2014): E924—E932. http://dx.doi.org/10.1073/pnas.1400236111.
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DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9D10A nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.
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Fernandes, Margret S., Mamatha M. Reddy, Jeffrey R. Gonneville, Scott C. DeRoo, Klaus Podar, James D. Griffin, David M. Weinstock, and Martin Sattler. "BCR-ABL promotes the frequency of mutagenic single-strand annealing DNA repair." Blood 114, no. 9 (August 27, 2009): 1813–19. http://dx.doi.org/10.1182/blood-2008-07-172148.
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Intracellular oxidative stress in cells transformed by the BCR-ABL oncogene is associated with increased DNA double-strand breaks. Imprecise repair of these breaks can result in the accumulation of mutations, leading to therapy-related drug resistance and disease progression. Using several BCR-ABL model systems, we found that BCR-ABL specifically promotes the repair of double-strand breaks through single-strand annealing (SSA), a mutagenic pathway that involves sequence repeats. Moreover, our results suggest that mutagenic SSA repair can be regulated through the interplay between BCR-ABL and extrinsic growth factors. Increased SSA activity required Y177 in BCR-ABL, as well as a functional PI3K and Ras pathway downstream of this site. Furthermore, our data hint at a common pathway for DSB repair whereby BCR-ABL, Tel-ABL, Tel-PDGFR, FLT3-ITD, and Jak2V617F all increase mutagenic repair. This increase in SSA may not be sufficiently suppressed by tyrosine kinase inhibitors in the stromal microenvironment. Therefore, drugs that target growth factor receptor signaling represent potential therapeutic agents to combat tyrosine kinase-induced genomic instability.
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An, Liwei, Chao Dong, Junshi Li, Jie Chen, Jingsong Yuan, Jun Huang, Kui Ming Chan, Cheng-han Yu, and Michael S. Y. Huen. "RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair." Proceedings of the National Academy of Sciences 115, no. 35 (August 13, 2018): E8286—E8295. http://dx.doi.org/10.1073/pnas.1804823115.
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Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.
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JOSEPH JERRY, D., NICHOLAS B. GRINER, and LUWEI TAO. "TUMOR SUPPRESSOR PATHWAYS AND CELLULAR ORIGINS OF BREAST CANCER: NEW COMPLEXITIES AND NEW HOPES." Nano LIFE 01, no. 01n02 (March 2010): 1–16. http://dx.doi.org/10.1142/s179398441000002x.
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Heritable breast cancer syndromes have identified the recognition and processing of DNA double strand breaks as a fundamental vulnerability in the breast epithelium. The role of homology-directed DNA repair is particularly prominent, indicating that this repair pathway is rate-limiting. Although the activities of the tumor suppressor genes underlying heritable breast cancer act in a common pathway of DNA double strand break repair, the specific lesions result in surprisingly different patterns of biomarkers in the breast cancers, suggesting that they arise from different cell types that include the luminal, basal and progenitor cells within the breast epithelium. Therefore, each cell type appears to have distinct underlying vulnerabilities in repair of DNA double strand breaks. While the heterogeneity of targets poses a challenge to develop specific therapies, these pathways also render tumor cells sensitive to drugs targeting double strand break repair pathways offering new options for therapies. As double strand break repair is a common pathway underlying breast cancer risk, therapies that enhance the proficiency of this pathway offer a strategy for chemoprevention.
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Weinstock, David M., and Maria Jasin. "Alternative Pathways for the Repair of RAG-Induced DNA Breaks." Molecular and Cellular Biology 26, no. 1 (January 1, 2006): 131–39. http://dx.doi.org/10.1128/mcb.26.1.131-139.2006.
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ABSTRACT RAG1 and RAG2 cleave DNA to generate blunt signal ends and hairpin coding ends at antigen receptor loci in lymphoid cells. During V(D)J recombination, repair of these RAG-generated double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway contributes substantially to the antigen receptor diversity necessary for immune system function, although recent evidence also supports the ability of RAG-generated breaks to undergo homology-directed repair (HDR). We have determined that RAG-generated chromosomal breaks can be repaired by pathways other than NHEJ in mouse embryonic stem (ES) cells, although repair by these pathways occurs at a significantly lower frequency than NHEJ. HDR frequency was estimated to be ≥40-fold lower than NHEJ frequency for both coding end and signal end reporters. Repair by single-strand annealing was estimated to occur at a comparable or lower frequency than HDR. As expected, V(D)J recombination was substantially impaired in cells deficient for the NHEJ components Ku70, XRCC4, and DNA-PKcs. Concomitant with decreased NHEJ, RAG-induced HDR was increased in each of the mutants, including cells lacking DNA-PKcs, which has been implicated in hairpin opening. HDR was increased to the largest extent in Ku70 − / − cells, implicating the Ku70/80 DNA end-binding protein in regulating pathway choice. Thus, RAG-generated DSBs are typically repaired by the NHEJ pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways in these cells.
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Kuzminov, Andrei. "Recombinational Repair of DNA Damage inEscherichia coli and Bacteriophage λ." Microbiology and Molecular Biology Reviews 63, no. 4 (December 1, 1999): 751–813. http://dx.doi.org/10.1128/mmbr.63.4.751-813.1999.
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SUMMARY Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.
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Yan, Hong, Jill McCane, Thomas Toczylowski, and Chinyi Chen. "Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair." Journal of Cell Biology 171, no. 2 (October 24, 2005): 217–27. http://dx.doi.org/10.1083/jcb.200502077.
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Werner syndrome is associated with premature aging and increased risk of cancer. Werner syndrome protein (WRN) is a RecQ-type DNA helicase, which seems to participate in DNA replication, double-strand break (DSB) repair, and telomere maintenance; however, its exact function remains elusive. Using Xenopus egg extracts as the model system, we found that Xenopus WRN (xWRN) is recruited to discrete foci upon induction of DSBs. Depletion of xWRN has no significant effect on nonhomologous end-joining of DSB ends, but it causes a significant reduction in the homology-dependent single-strand annealing DSB repair pathway. These results provide the first direct biochemical evidence that links WRN to a specific DSB repair pathway. The assay for single-strand annealing that was developed in this study also provides a powerful biochemical system for mechanistic analysis of homology-dependent DSB repair.
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Summers, K. C., F. Shen, E. A. Sierra Potchanant, E. A. Phipps, R. J. Hickey, and L. H. Malkas. "Phosphorylation: The Molecular Switch of Double-Strand Break Repair." International Journal of Proteomics 2011 (May 18, 2011): 1–8. http://dx.doi.org/10.1155/2011/373816.
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Repair of double-stranded breaks (DSBs) is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ), homologous recombination (HR), or the inclusive DNA damage response (DDR). These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.
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Xu, Yixi, and Dongyi Xu. "Repair pathway choice for double-strand breaks." Essays in Biochemistry 64, no. 5 (July 10, 2020): 765–77. http://dx.doi.org/10.1042/ebc20200007.
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Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.
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Wynn, Emily, Emma Purfeerst, and Alan Christensen. "Mitochondrial DNA Repair in an Arabidopsis thaliana Uracil N-Glycosylase Mutant." Plants 9, no. 2 (February 18, 2020): 261. http://dx.doi.org/10.3390/plants9020261.
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Substitution rates in plant mitochondrial genes are extremely low, indicating strong selective pressure as well as efficient repair. Plant mitochondria possess base excision repair pathways; however, many repair pathways such as nucleotide excision repair and mismatch repair appear to be absent. In the absence of these pathways, many DNA lesions must be repaired by a different mechanism. To test the hypothesis that double-strand break repair (DSBR) is that mechanism, we maintained independent self-crossing lineages of plants deficient in uracil-N-glycosylase (UNG) for 11 generations to determine the repair outcomes when that pathway is missing. Surprisingly, no single nucleotide polymorphisms (SNPs) were fixed in any line in generation 11. The pattern of heteroplasmic SNPs was also unaltered through 11 generations. When the rate of cytosine deamination was increased by mitochondrial expression of the cytosine deaminase APOBEC3G, there was an increase in heteroplasmic SNPs but only in mature leaves. Clearly, DNA maintenance in reproductive meristem mitochondria is very effective in the absence of UNG while mitochondrial genomes in differentiated tissue are maintained through a different mechanism or not at all. Several genes involved in DSBR are upregulated in the absence of UNG, indicating that double-strand break repair is a general system of repair in plant mitochondria. It is important to note that the developmental stage of tissues is critically important for these types of experiments.
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Alekseev, Aleksandr, Galina Cherevatenko, Maksim Serdakov, Georgii Pobegalov, Alexander Yakimov, Irina Bakhlanova, Dmitry Baitin, and Mikhail Khodorkovskii. "Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA." International Journal of Molecular Sciences 21, no. 19 (October 7, 2020): 7389. http://dx.doi.org/10.3390/ijms21197389.
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Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.
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Dilworth, David, Fade Gong, Kyle Miller, and Christopher J. Nelson. "FKBP25 participates in DNA double-strand break repair." Biochemistry and Cell Biology 98, no. 1 (February 2020): 42–49. http://dx.doi.org/10.1139/bcb-2018-0328.
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FK506-binding proteins (FKBPs) alter the conformation of proteins via cis–trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25’s catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.
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Frigerio, Chiara, Elena Di Nisio, Michela Galli, Chiara Vittoria Colombo, Rodolfo Negri, and Michela Clerici. "The Chromatin Landscape around DNA Double-Strand Breaks in Yeast and Its Influence on DNA Repair Pathway Choice." International Journal of Molecular Sciences 24, no. 4 (February 7, 2023): 3248. http://dx.doi.org/10.3390/ijms24043248.
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DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.
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Vasil'eva, M., A. Bugay, and E. Dushanov. "MODELING OF DNA DAMAGE REPAIR INDUCED BY HEAVY IONS IN MAMMALIAN CELLS." Russian Journal of Biological Physics and Chemisrty 7, no. 4 (November 24, 2022): 557–64. http://dx.doi.org/10.29039/rusjbpc.2022.0560.
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In this paper the mathematical description of main DNA repair pathways of single-strand break (SSB), base damage (BD), and double-strand break (DSB) in mammalian and human cells are proposed. The model shows key molecular mechanisms of DNA recovery through the single-strand DNA repair, base excision repair (BER), nonhomologous end-joining (NHEJ). To formalize the molecular mechanisms the dynamic system of differential equations describing the chemical kinetics of protein interactions according the modern concepts of molecular biology is constructed. Taking into account three repair pathways it makes possible to describe the cell's response to heavy charged particles influence. The proposed model is validated for main mechanisms of SSB repair, BER, NHEJ. In the course of the work, the time-dependent dynamics of formations and repairs of key DNA damage types (BD, SSB, DSB, cluster damages) in human cells under 56Fe ions (E = 600 Mev/u) exposure are calculated. A comparative analysis of the DNA damages and theirs repair under 12C (E = 270 MeV/u) and 56Fe (E = 600 Mev/u) ions exposure at 1 Gy was carried out.
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Zhao, Fei, Wootae Kim, Jake A. Kloeber, and Zhenkun Lou. "DNA end resection and its role in DNA replication and DSB repair choice in mammalian cells." Experimental & Molecular Medicine 52, no. 10 (October 2020): 1705–14. http://dx.doi.org/10.1038/s12276-020-00519-1.
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Abstract DNA end resection has a key role in double-strand break repair and DNA replication. Defective DNA end resection can cause malfunctions in DNA repair and replication, leading to greater genomic instability. DNA end resection is initiated by MRN-CtIP generating short, 3′-single-stranded DNA (ssDNA). This newly generated ssDNA is further elongated by multiple nucleases and DNA helicases, such as EXO1, DNA2, and BLM. Effective DNA end resection is essential for error-free homologous recombination DNA repair, the degradation of incorrectly replicated DNA and double-strand break repair choice. Because of its importance in DNA repair, DNA end resection is strictly regulated. Numerous mechanisms have been reported to regulate the initiation, extension, and termination of DNA end resection. Here, we review the general process of DNA end resection and its role in DNA replication and repair pathway choice.
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Gupta, Richa, Stewart Shuman, and Michael S. Glickman. "RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria." Journal of Bacteriology 197, no. 19 (July 20, 2015): 3121–32. http://dx.doi.org/10.1128/jb.00290-15.
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ABSTRACTMycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation ofadnABorrecOindividually causes partial impairment of HR, but loss ofadnABandrecOin combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNAin vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis ofrecFandrecRin mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCEThis study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284–2295, 2013,http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.
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Daley, James M., and Thomas E. Wilson. "Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length." Molecular and Cellular Biology 25, no. 3 (February 1, 2005): 896–906. http://dx.doi.org/10.1128/mcb.25.3.896-906.2005.
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ABSTRACT The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.
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Hooshyar, Mohsen, Daniel Burnside, Maryam Hajikarimlou, Katayoun Omidi, Alexander Jesso, Megan Vanstone, Adamo Young, et al. "Actin-Related Protein 6 (Arp6) Influences Double-Strand Break Repair in Yeast." Applied Microbiology 1, no. 2 (July 16, 2021): 225–38. http://dx.doi.org/10.3390/applmicrobiol1020017.
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DNA double-strand breaks (DSBs) are the most deleterious form of DNA damage and are repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair initiation, regulation and communication with signaling pathways require several histone-modifying and chromatin-remodeling complexes. In budding yeast, this involves three primary complexes: INO80-C, which is primarily associated with HR, SWR1-C, which promotes NHEJ, and RSC-C, which is involved in both pathways as well as the general DNA damage response. Here we identify ARP6 as a factor involved in DSB repair through an RSC-C-related pathway. The loss of ARP6 significantly reduces the NHEJ repair efficiency of linearized plasmids with cohesive ends, impairs the repair of chromosomal breaks, and sensitizes cells to DNA-damaging agents. Genetic interaction analysis indicates that ARP6, MRE11 and RSC-C function within the same pathway, and the overexpression of ARP6 rescues rsc2∆ and mre11∆ sensitivity to DNA-damaging agents. Double mutants of ARP6, and members of the INO80 and SWR1 complexes, cause a significant reduction in repair efficiency, suggesting that ARP6 functions independently of SWR1-C and INO80-C. These findings support a novel role for ARP6 in DSB repair that is independent of the SWR1 chromatin remodeling complex, through an apparent RSC-C and MRE11-associated DNA repair pathway.
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Nick McElhinny, Stephanie A., and Dale A. Ramsden. "Polymerase Mu Is a DNA-Directed DNA/RNA Polymerase." Molecular and Cellular Biology 23, no. 7 (April 1, 2003): 2309–15. http://dx.doi.org/10.1128/mcb.23.7.2309-2315.2003.
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ABSTRACT DNA polymerases are defined as such because they use deoxynucleotides instead of ribonucleotides with high specificity. We show here that polymerase mu (pol μ), implicated in the nonhomologous end-joining pathway for repair of DNA double-strand breaks, incorporates both ribonucleotides and deoxynucleotides in a template-directed manner. pol μ has an approximately 1,000-fold-reduced ability to discriminate against ribonucleotides compared to that of the related pol β, although pol μ's substrate specificity is similar to that of pol β in most other respects. Moreover, pol μ more frequently incorporates ribonucleotides when presented with nucleotide concentrations that approximate cellular pools. We therefore addressed the impact of ribonucleotide incorporation on the activities of factors required for double-strand break repair by nonhomologous end joining. We determined that the ligase required for this pathway readily joined strand breaks with terminal ribonucleotides. Most significantly, pol μ frequently introduced ribonucleotides into the repair junctions of an in vitro nonhomologous end-joining reaction, an activity that would be expected to have important consequences in the context of cellular double-strand break repair.
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IJff, Marloes, Gregor G. W. van Bochove, Denise Whitton, Roy Winiarczyk, Celina Honhoff, Hans Rodermond, Johannes Crezee, Lukas J. A. Stalpers, Nicolaas A. P. Franken, and Arlene L. Oei. "PARP1-Inhibition Sensitizes Cervical Cancer Cell Lines for Chemoradiation and Thermoradiation." Cancers 13, no. 9 (April 26, 2021): 2092. http://dx.doi.org/10.3390/cancers13092092.
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Radiotherapy plus cisplatin (chemoradiation) is standard treatment for women with locoregionally advanced cervical cancer. Both radiotherapy and cisplatin induce DNA single and double-strand breaks (SSBs and DSBs). These double-strand breaks can be repaired via two major DNA repair pathways: Classical Non-Homologous End-Joining (cNHEJ) and Homologous Recombination. Besides inducing DNA breaks, cisplatin also disrupts the cNHEJ pathway. Patients contra-indicated for cisplatin are treated with radiotherapy plus hyperthermia (thermoradiation). Hyperthermia inhibits the HR pathway. The aim of our study is to enhance chemoradiation or thermoradiation by adding PARP1-inhibition, which disrupts both the SSB repair and the Alternative NHEJ DSB repair pathway. This was studied in cervical cancer cell lines (SiHa, HeLa, C33A and CaSki) treated with hyperthermia (42 °C) ± ionizing radiation (2–6 Gy) ± cisplatin (0.3–0.5 µM) ± PARP1-inhibitor (olaparib, 4.0–5.0 µM). Clonogenic assays were performed to measure cell reproductive death. DSBs were analyzed by γ-H2AX staining and cell death by live cell imaging. Both chemoradiation and thermoradiation resulted in lower survival fractions and increased unrepaired DSBs when combined with a PARP1-inhibitor. A quadruple modality, including ionizing radiation, hyperthermia, cisplatin and PARP1-i, was not more effective than either triple modality. However, both chemoradiation and thermoradiation benefit significantly from additional treatment with PARP1-i.
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Hossain, Md, Yunfeng Lin, and Shan Yan. "Single-Strand Break End Resection in Genome Integrity: Mechanism and Regulation by APE2." International Journal of Molecular Sciences 19, no. 8 (August 14, 2018): 2389. http://dx.doi.org/10.3390/ijms19082389.
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DNA single-strand breaks (SSBs) occur more than 10,000 times per mammalian cell each day, representing the most common type of DNA damage. Unrepaired SSBs compromise DNA replication and transcription programs, leading to genome instability. Unrepaired SSBs are associated with diseases such as cancer and neurodegenerative disorders. Although canonical SSB repair pathway is activated to repair most SSBs, it remains unclear whether and how unrepaired SSBs are sensed and signaled. In this review, we propose a new concept of SSB end resection for genome integrity. We propose a four-step mechanism of SSB end resection: SSB end sensing and processing, as well as initiation, continuation, and termination of SSB end resection. We also compare different mechanisms of SSB end resection and DSB end resection in DNA repair and DNA damage response (DDR) pathways. We further discuss how SSB end resection contributes to SSB signaling and repair. We focus on the mechanism and regulation by APE2 in SSB end resection in genome integrity. Finally, we identify areas of future study that may help us gain further mechanistic insight into the process of SSB end resection. Overall, this review provides the first comprehensive perspective on SSB end resection in genome integrity.
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Tijsterman, Marcel, Remko de Pril, Judith G. Tasseron-de Jong, and Jaap Brouwer. "RNA Polymerase II Transcription Suppresses Nucleosomal Modulation of UV-Induced (6-4) Photoproduct and Cyclobutane Pyrimidine Dimer Repair in Yeast." Molecular and Cellular Biology 19, no. 1 (January 1, 1999): 934–40. http://dx.doi.org/10.1128/mcb.19.1.934.
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ABSTRACT The nucleotide excision repair (NER) pathway is able to remove a wide variety of structurally unrelated lesions from DNA. NER operates throughout the genome, but the efficiencies of lesion removal are not the same for different genomic regions. Even within a single gene or DNA strand repair rates vary, and this intragenic heterogeneity is of considerable interest with respect to the mutagenic potential of carcinogens. In this study, we have analyzed the removal of the two major types of genotoxic DNA adducts induced by UV light, i.e., the pyrimidine (6-4)-pyrimidone photoproduct (6-4PP) and the cyclobutane pyrimidine dimer (CPD), from the Saccharomyces cerevisiae URA3 gene at nucleotide resolution. In contrast to the fast and uniform removal of CPDs from the transcribed strand, removal of lesions from the nontranscribed strand is generally less efficient and is modulated by the chromatin environment of the damage. Removal of 6-4PPs from nontranscribed sequences is also profoundly influenced by positioned nucleosomes, but this type of lesion is repaired at a much higher rate. Still, the transcribed strand is repaired preferentially, indicating that, as in the removal of CPDs, transcription-coupled repair predominates in the removal of 6-4PPs from transcribed DNA. The hypothesis that transcription machinery operates as the rate-determining damage recognition entity in transcription-coupled repair is supported by the observation that this pathway removes both types of UV photoproducts at equal rates without being profoundly influenced by the sequence or chromatin context.
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Davis, Allison P., and Lorraine S. Symington. "The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing." Genetics 159, no. 2 (October 1, 2001): 515–25. http://dx.doi.org/10.1093/genetics/159.2.515.
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Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.
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West, C. E., W. M. Waterworth, P. A. Sunderland, and C. M. Bray. "Arabidopsis DNA double-strand break repair pathways." Biochemical Society Transactions 32, no. 6 (October 26, 2004): 964–66. http://dx.doi.org/10.1042/bst0320964.
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DSBs (double-strand breaks) are one of the most serious forms of DNA damage that can occur in a cell's genome. DNA replication in cells containing DSBs, or following incorrect repair, may result in the loss of large amounts of genetic material, aneuploid daughter cells and cell death. There are two major pathways for DSB repair: HR (homologous recombination) uses an intact copy of the damaged region as a template for repair, whereas NHEJ (non-homologous end-joining) rejoins DNA ends independently of DNA sequence. In most plants, NHEJ is the predominant DSB repair pathway. Previously, the Arabidopsis NHEJ mutant atku80 was isolated and found to display hypersensitivity to bleomycin, a drug that causes DSBs in DNA. In the present study, the transcript profiles of wild-type and atku80 mutant plants grown in the presence and absence of bleomycin are determined by microarray analysis. Several genes displayed very strong transcriptional induction specifically in response to DNA damage, including the characterized DSB repair genes AtRAD51 and AtBRCA1. These results identify novel candidate genes that encode components of the DSB repair pathways active in NHEJ mutant plants.
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Cejka, Petr, and Lorraine S. Symington. "DNA End Resection: Mechanism and Control." Annual Review of Genetics 55, no. 1 (November 23, 2021): 285–307. http://dx.doi.org/10.1146/annurev-genet-071719-020312.
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DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5′→3′ nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.
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Cook, Diana, Sarah Long, John Stanton, Patrick Cusick, Colleen Lawrimore, Elaine Yeh, Sarah Grant, and Kerry Bloom. "Behavior of dicentric chromosomes in budding yeast." PLOS Genetics 17, no. 3 (March 18, 2021): e1009442. http://dx.doi.org/10.1371/journal.pgen.1009442.
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DNA double-strand breaks arisein vivowhen a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.
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Gangavarapu, Venkateswarlu, Satya Prakash, and Louise Prakash. "Requirement of RAD52 Group Genes for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae." Molecular and Cellular Biology 27, no. 21 (September 4, 2007): 7758–64. http://dx.doi.org/10.1128/mcb.01331-07.
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ABSTRACT In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases η and ζ and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.
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Moggs, Jonathan G., Paola Grandi, Jean-Pierre Quivy, Zophonías O. Jónsson, Ulrich Hübscher, Peter B. Becker, and Geneviève Almouzni. "A CAF-1–PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage." Molecular and Cellular Biology 20, no. 4 (February 15, 2000): 1206–18. http://dx.doi.org/10.1128/mcb.20.4.1206-1218.2000.
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ABSTRACT Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control.
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Mladenov, Emil, Christian Staudt, Aashish Soni, Tamara Murmann-Konda, Maria Siemann-Loekes, and George Iliakis. "Strong suppression of gene conversion with increasing DNA double-strand break load delimited by 53BP1 and RAD52." Nucleic Acids Research 48, no. 4 (December 13, 2019): 1905–24. http://dx.doi.org/10.1093/nar/gkz1167.
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Abstract In vertebrates, genomic DNA double-strand breaks (DSBs) are removed by non-homologous end-joining processes: classical non-homologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ); or by homology-dependent processes: gene-conversion (GC) and single-strand annealing (SSA). Surprisingly, these repair pathways are not real alternative options restoring genome integrity with equal efficiency, but show instead striking differences in speed, accuracy and cell-cycle-phase dependence. As a consequence, engagement of one pathway may be associated with processing-risks for the genome absent from another pathway. Characterization of engagement-parameters and their consequences is, therefore, essential for understanding effects on the genome of DSB-inducing agents, such as ionizing-radiation (IR). Here, by addressing pathway selection in G2-phase, we discover regulatory confinements in GC with consequences for SSA- and c-NHEJ-engagement. We show pronounced suppression of GC with increasing DSB-load that is not due to RAD51 availability and which is delimited but not defined by 53BP1 and RAD52. Strikingly, at low DSB-loads, GC repairs ∼50% of DSBs, whereas at high DSB-loads its contribution is undetectable. Notably, with increasing DSB-load and the associated suppression of GC, SSA gains ground, while alt-EJ is suppressed. These observations explain earlier, apparently contradictory results and advance our understanding of logic and mechanisms underpinning the wiring between DSB repair pathways.
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Murray, Johanne M., Tom Stiff, and Penny A. Jeggo. "DNA double-strand break repair within heterochromatic regions." Biochemical Society Transactions 40, no. 1 (January 19, 2012): 173–78. http://dx.doi.org/10.1042/bst20110631.
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DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.
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Rodrigues, Margret S., Jeffrey R. Gonneville, Klaus Podar, David M. Weinstock, James D. Griffin, and Martin Sattler. "BCR-ABL Induces Error-Prone Single Strand Annealing in Transformed Cells." Blood 110, no. 11 (November 16, 2007): 2937. http://dx.doi.org/10.1182/blood.v110.11.2937.2937.
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Abstract Chronic myelogenous leukemia (CML) is a hematopoietic stem cell disorder, caused by the BCR-ABL tyrosine kinase oncogene. BCR-ABL kinase activity is required for all aspects of transformation, including abnormal proliferation and neoplastic expansion of stem cells, activation of signaling pathways and genomic instability. We have recently shown that BCR-ABL kinase activity also causes elevated intracellular levels of reactive oxygen species (ROS). These chronically elevated ROS levels have been implicated in the induction of DNA double-strand breaks (DSB) and therapy-related drug resistance, mainly through low- fidelity homology-directed repair (HDR) of DNA lesions. We have utilized GFP-based reporters in BCR-ABL transformed cells to measure both HDR and single-strand annealing (SSA), a mutagenic pathway of homologous repair between repetitive sequences. Repair rates of a single DSB by each of these pathways was measured in BCR-ABL expressing BaF3 cells (BaF3.p210), which have previously been shown to be an excellent system to model the induction of imatinib resistant mutations. We found that inhibition of BCR-ABL kinase activity by imatinib decreased the frequency of SSA by 73 ± 2% (n=3) and increased the frequency of HDR by 70 ± 11% (n=3) in BaF3.p210, compared to untreated cells. Treatment with imatinib did not affect HDR or SSA in BaF3 cells that do not express BCR-ABL. We also determined the fidelity of both SSA and HDR in our model system using clonal populations that stably express the repaired GFP+ reporters (n=20). As expected, sequencing of GFP+ repair products from cells containing the SSA reporter confirmed the expected sequence deletions, consistent with an error-prone mechanism. In contrast, sequencing of GFP+ repair products from the HDR reporter indicated a high-fidelity repair mechanism, without any mutations. It should be noted that this reporter assay does not account for potentially imprecise HDR, leading to a non-functional GFP. Nevertheless, point mutations at the previously reported rate were not detected. Thus, altered HDR fidelity may not be a universal mechanism for the induction of point mutations by BCR-ABL. Our data suggests a unique and alternative pathway, whereby BCR-ABL alters the balance between the SSA and HDR pathways. Moreover, in the presence of interleukin-3 at a concentration that supports not only viability but also cell growth (1ng/ml), imatinib was ineffective at protecting cells from error-prone SSA. In vivo, stromal cells may provide these additional growth signals. Our in vitro data show that stromal cell conditioned medium is not only sufficient to support growth and viability of CML cell lines in the presence of imatinib, but it can also lead to elevated levels of ROS. An abnormal increase in ROS is sufficient by itself to cause genomic mutations (transitions and/or transversions) as a result of single strand oxidative DNA lesions, even in the absence of altered DSB or single strand lesion repair mechanisms. Thus, therapy-related resistance and additional genomic abnormalities may not only be a result of BCR-ABL dependent ROS induction but may also occur within the stromal cell microenvironment through ROS induction by growth factors.
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Burgess, Joshua T., Chee Man Cheong, Amila Suraweera, Thais Sobanski, Sam Beard, Keyur Dave, Maddison Rose, et al. "Barrier-to-autointegration-factor (Banf1) modulates DNA double-strand break repair pathway choice via regulation of DNA-dependent kinase (DNA-PK) activity." Nucleic Acids Research 49, no. 6 (February 28, 2021): 3294–307. http://dx.doi.org/10.1093/nar/gkab110.
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AbstractDNA repair pathways are essential to maintain the integrity of the genome and prevent cell death and tumourigenesis. Here, we show that the Barrier-to-Autointegration Factor (Banf1) protein has a role in the repair of DNA double-strand breaks. Banf1 is characterized as a nuclear envelope protein and mutations in Banf1 are associated with the severe premature aging syndrome, Néstor–Guillermo Progeria Syndrome. We have previously shown that Banf1 directly regulates the activity of PARP1 in the repair of oxidative DNA lesions. Here, we show that Banf1 also has a role in modulating DNA double-strand break repair through regulation of the DNA-dependent Protein Kinase catalytic subunit, DNA-PKcs. Specifically, we demonstrate that Banf1 relocalizes from the nuclear envelope to sites of DNA double-strand breaks. We also show that Banf1 can bind to and directly inhibit the activity of DNA-PKcs. Supporting this, cellular depletion of Banf1 leads to an increase in non-homologous end-joining and a decrease in homologous recombination, which our data suggest is likely due to unrestrained DNA-PKcs activity. Overall, this study identifies how Banf1 regulates double-strand break repair pathway choice by modulating DNA-PKcs activity to control genome stability within the cell.
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Krwawicz, Joanna, Katarzyna D. Arczewska, Elzbieta Speina, Agnieszka Maciejewska, and Elzbieta Grzesiuk. "Bacterial DNA repair genes and their eukaryotic homologues: 1. Mutations in genes involved in base excision repair (BER) and DNA-end processors and their implication in mutagenesis and human disease." Acta Biochimica Polonica 54, no. 3 (September 24, 2007): 413–34. http://dx.doi.org/10.18388/abp.2007_3219.
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Base excision repair (BER) pathway executed by a complex network of proteins is the major system responsible for the removal of damaged DNA bases and repair of DNA single strand breaks (SSBs) generated by environmental agents, such as certain cancer therapies, or arising spontaneously during cellular metabolism. Both modified DNA bases and SSBs with ends other than 3'-OH and 5'-P are repaired either by replacement of a single or of more nucleotides in the processes called short-patch BER (SP-BER) or long-patch BER (LP-BER), respectively. In contrast to Escherichia coli cells, in human ones, the two BER sub-pathways are operated by different sets of proteins. In this review the selection between SP- and LP-BER and mutations in BER and end-processors genes and their contribution to bacterial mutagenesis and human diseases are considered.
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Yokochi, T., K. Kusano, and I. Kobayashi. "Evidence for conservative (two-progeny) DNA double-strand break repair." Genetics 139, no. 1 (January 1, 1995): 5–17. http://dx.doi.org/10.1093/genetics/139.1.5.
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Abstract The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage lambda Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.
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XU, DongYi, and HuaYu ZHAO. "Pathway choice for DNA double strand break repair." SCIENTIA SINICA Vitae 51, no. 1 (November 25, 2020): 56–69. http://dx.doi.org/10.1360/ssv-2020-0196.
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Stahl-Rommel, Sarah, David Li, Michelle Sung, Rebecca Li, Aarthi Vijayakumar, Kutay Deniz Atabay, G. Guy Bushkin, et al. "A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station." PLOS ONE 16, no. 6 (June 30, 2021): e0253403. http://dx.doi.org/10.1371/journal.pone.0253403.
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As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station.
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Shaheen, Montaser, Christopher Allen, Jac A. Nickoloff, and Robert Hromas. "Synthetic lethality: exploiting the addiction of cancer to DNA repair." Blood 117, no. 23 (June 9, 2011): 6074–82. http://dx.doi.org/10.1182/blood-2011-01-313734.
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Abstract Because cancer at its origin must acquire permanent genomic mutations, it is by definition a disease of DNA repair. Yet for cancer cells to replicate their DNA and divide, which is the fundamental phenotype of cancer, multiple DNA repair pathways are required. This produces a paradox for the cancer cell, where its origin is at the same time its weakness. To overcome this difficulty, a cancer cell often becomes addicted to DNA repair pathways other than the one that led to its initial mutability. The best example of this is in breast or ovarian cancers with mutated BRCA1 or 2, essential components of a repair pathway for repairing DNA double-strand breaks. Because replicating DNA requires repair of DNA double-strand breaks, these cancers have become reliant on another DNA repair component, PARP1, for replication fork progression. The inhibition of PARP1 in these cells results in catastrophic double-strand breaks during replication, and ultimately cell death. The exploitation of the addiction of cancer cells to a DNA repair pathway is based on synthetic lethality and has wide applicability to the treatment of many types of malignancies, including those of hematologic origin. There is a large number of novel compounds in clinical trials that use this mechanism for their antineoplastic activity, making synthetic lethality one of the most important new concepts in recent drug development.
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Davis, Destiny K., Gabrielle Elliott, Lindsay Renshaw, and Emily Moser. "The role of the ubiquitin ligase Cul4b in B lymphocytes." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 154.18. http://dx.doi.org/10.4049/jimmunol.210.supp.154.18.
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Abstract Vaccination or infection induces antibody secretion by long-lived plasma cells, creating durable immune protection against secondary infections. These plasma cells develop from B cells that have undergone affinity maturation, which requires somatic hypermutation in proliferating B cells, forming single strand DNA breaks that are repaired by mismatch repair machinery. Cullin 4b (Cul4b) is an E3 ubiquitin ligase found in most cells, including B cells, but its function in B cells is unknown. Cul4b is one of two docking proteins forming the backbone of the Cullin RING Ligase 4 (CRL4) complex, which ubiquitinates substrate proteins for degradation. Previous studies have shown that Cul4b promotes DNA repair with cell proliferation, and high Cul4b is linked to aggressive colon cancer. Because proliferation and DNA repair are essential for humoral immunity, we hypothesized that Cul4b also functions in B cells. To test this, we generated mice that lack Cul4b in B cells. We immunized these mice with an mRNA vaccine against an H1N1 Influenza A virus, and measured virus-specific serum antibody. We found that Influenza-specific antibodies were dramatically reduced in mice serum that lacked Cul4b in B cells. To determine if Cul4b promotes DNA damage repair in B cells, we induced single-strand DNA breaks with UV irradiation and then stimulated the cells to proliferate in vitro. We found that Cul4b promoted B lymphocyte proliferation after UV exposure. Understanding the Cul4b-DNA damage repair pathway in B cells will identify new drug targets to transiently amplify this pathway to improve vaccines and encourage stronger antibody responses, or to inhibit this pathway to prevent tumor progression in cancer.
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Campalans, Anna, Eva Moritz, Thierry Kortulewski, Denis Biard, Bernd Epe, and J. Pablo Radicella. "Interaction with OGG1 Is Required for Efficient Recruitment of XRCC1 to Base Excision Repair and Maintenance of Genetic Stability after Exposure to Oxidative Stress." Molecular and Cellular Biology 35, no. 9 (March 2, 2015): 1648–58. http://dx.doi.org/10.1128/mcb.00134-15.
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XRCC1 is an essential protein required for the maintenance of genomic stability through its implication in DNA repair. The main function of XRCC1 is associated with its role in the single-strand break (SSB) and base excision repair (BER) pathways that share several enzymatic steps. We show here that the polymorphic XRCC1 variant R194W presents a defect in its interaction with the DNA glycosylase OGG1 after oxidative stress. While proficient for single-strand break repair (SSBR), this variant does not colocalize with OGG1, reflecting a defect in its involvement in BER. Consistent with a role of XRCC1 in the coordination of the BER pathway, induction of oxidative base damage in XRCC1-deficient cells complemented with the R194W variant results in increased genetic instability as revealed by the accumulation of micronuclei. These data identify a specific molecular role for the XRCC1-OGG1 interaction in BER and provide a model for the effects of the R194W variant identified in molecular cancer epidemiology studies.
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Heaton, Brook E., Daniel Barkan, Paola Bongiorno, Petros C. Karakousis, and Michael S. Glickman. "Deficiency of Double-Strand DNA Break Repair Does Not Impair Mycobacterium tuberculosis Virulence in Multiple Animal Models of Infection." Infection and Immunity 82, no. 8 (May 19, 2014): 3177–85. http://dx.doi.org/10.1128/iai.01540-14.
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ABSTRACTMycobacterium tuberculosispersistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, includingM. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed.M. tuberculosiselaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant toM. tuberculosislatency. However, very little information is available about the phenotype of DSB repair-deficientM. tuberculosisin animal models of infection. Here we testedM. tuberculosisstrains lacking NHEJ (a ΔkuΔligDstrain), HR (a ΔrecAstrain), or both (a ΔrecAΔkustrain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required forM. tuberculosispathogenesis.
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Manukyan, A. T. "The Activation оf DNА Repair Pathways after Ultra-Short Pulsed Electron Beam Irradiation in Human Cells." Reports of NAS RA 122, no. 2 (2022): 161–66. http://dx.doi.org/10.54503/0321-1339-2022.122.2-161.
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The aim of this work was to estimate the differences in activation of DNA repair pathways of DNA double-strand and single-strand breaks induced by ultrashort pulse electron beam irradiation in human K-562 cells. The activation of HR, NHEJ and BER DNA repair pathways was studied at non-lethal, sub-lethal and lethal doses of irradiation. Our results indicate that the activation of the specific repair pathways and repair kinetics depend on the irradiation dose.
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Aquila, Lanni, and Boyko S. Atanassov. "Regulation of Histone Ubiquitination in Response to DNA Double Strand Breaks." Cells 9, no. 7 (July 16, 2020): 1699. http://dx.doi.org/10.3390/cells9071699.
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Eukaryotic cells are constantly exposed to both endogenous and exogenous stressors that promote the induction of DNA damage. Of this damage, double strand breaks (DSBs) are the most lethal and must be efficiently repaired in order to maintain genomic integrity. Repair of DSBs occurs primarily through one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these pathways is in part regulated by histone post-translational modifications (PTMs) including ubiquitination. Ubiquitinated histones not only influence transcription and chromatin architecture at sites neighboring DSBs but serve as critical recruitment platforms for repair machinery as well. The reversal of these modifications by deubiquitinating enzymes (DUBs) is increasingly being recognized in a number of cellular processes including DSB repair. In this context, DUBs ensure proper levels of ubiquitin, regulate recruitment of downstream effectors, dictate repair pathway choice, and facilitate appropriate termination of the repair response. This review outlines the current understanding of histone ubiquitination in response to DSBs, followed by a comprehensive overview of the DUBs that catalyze the removal of these marks.
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Oksenych, Valentyn. "DNA Repair and Immune Response: Editorial." Biomolecules 13, no. 1 (December 30, 2022): 84. http://dx.doi.org/10.3390/biom13010084.
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Ivanov, Evgeny L., Neal Sugawara, Jacqueline Fishman-Lobell, and James E. Haber. "Genetic Requirements for the Single-Strand Annealing Pathway of Double-Strand Break Repair in Saccharomyces cerevisiae." Genetics 142, no. 3 (March 1, 1996): 693–704. http://dx.doi.org/10.1093/genetics/142.3.693.
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Abstract HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching.