Academic literature on the topic 'Recombination Repair Pathway'
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Journal articles on the topic "Recombination Repair Pathway"
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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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Kang, Josephine, and Martin J. Blaser. "Repair and Antirepair DNA Helicases in Helicobacter pylori." Journal of Bacteriology 190, no. 12 (March 28, 2008): 4218–24. http://dx.doi.org/10.1128/jb.01848-07.
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ABSTRACT Orthologs of RecG and RuvABC are highly conserved among prokaryotes; in Escherichia coli, they participate in independent pathways that branch migrate Holliday junctions during recombinational DNA repair. RecG also has been shown to directly convert stalled replication forks into Holliday junctions. The bacterium Helicobacter pylori, with remarkably high levels of recombination, possesses RecG and RuvABC homologs, but in contrast to E. coli, H. pylori RecG limits recombinational repair. We now show that the RuvABC pathway plays the prominent, if not exclusive, repair role. By introducing an E. coli resolvase (RusA) into H. pylori, the repair and recombination phenotypes of the ruvB mutant but not the recG mutant were improved. Our results indicate that RecG and RuvB compete for Holliday junction structures in recombinational repair, but since a classic RecG resolvase is absent from H. pylori, deployment of the RecG pathway is lethal. We propose that evolutionary loss of the H. pylori RecG resolvase provides an “antirepair” pathway allowing for selection of varied strains. Such competition between repair and antirepair provides a novel mechanism to maximize fitness at a bacterial population level.
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Rocha, Pedro P., Yi Fu, JungHyun Kim, and Jane Skok. "The Impact of Nuclear Organization and Homolgous Recombination in Repair of DNA Damage Introduced By Aid during Class Switch Recombination." Blood 124, no. 21 (December 6, 2014): 2738. http://dx.doi.org/10.1182/blood.v124.21.2738.2738.
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Abstract Class Switch Recombination (CSR) involves the introduction of double stranded breaks (DSBs) at the switch regions of the immunoglulin heavy chain (Igh) locus by the enzyme Activation Cytidine Deaminse (AID). AID can also act as a general mutator targeting other loci in the genome which can then either be repaired faithfully or in an error-prone fashion introducing mutations and potentially initiating B cell lymphoma. The factors contributing to the choice of repair pathway are not fully understood. Here we tested the hypothesis that repair pathway choice is influenced by differential accessibility and expression levels of target loci across cell cycle. More specifically in the context of CSR we tested whether differential regulation of gene accessibility across cell cycle is an important determinant for AID binding and subsequent repair pathway choice as different repair pathways predominate at different stages of cell cycle. Using 3D-FISH in conjunction with Immunofluorescence we observed that AID target genes that are faithfully repaired are more accessible (found in euchromatic regions) in the G2 phase of the cell cycle then genes that are frequently mutated. In contrast, those genes which are repaired in an error prone fashion are more accessible in the G1 phase of cell cycle. Since Homologous Recombination mediated repair (HR), which is a faithful repair mechanism, occurs in G2 we speculate that accessibility of these genes at this stage of cell cycle facilitates action by this repair pathway. Conversely, genes that are more accessible during the G1 phase of cell cycle will be repaired by the non-homologous end joining (NHEJ) repair pathway and therefore are more likely to be mutated. Thus, HR could be the pathway by which faithful repair is accomplished and use of the NHEJ pathway on the other hand could contribute to the introduction of dangerous DNA mutations that might lead to B cell transformation and cancer. To connect differences in accessibility with repair pathway usage, we used a mouse model carrying a hypomorphic mutation in BRCA2, a protein involved in HR. This is the first mouse model impaired in HR that eludes embryonic lethality and allows inspection of the role of this pathway in maintaining genomic stability in splenocytes undergoing CSR. Our preliminary investigations indicate that in Brca2 mutant B cells not only is the integrity of fathfully repaired loci compromised, but the Igh locus is also damaged. Taken together these results support our hypothesis and further indicate that the HR pathway is involved in repairing Igh. Given that approximately 95% of lymphomas are of B cell origin and many of these are associated with AID mediated breaks, it is crucial for us to understand the factors that influence targeting and repair. Disclosures No relevant conflicts of interest to declare.
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Mendonca, V. M., and S. W. Matson. "Genetic analysis of delta helD and delta uvrD mutations in combination with other genes in the RecF recombination pathway in Escherichia coli: suppression of a ruvB mutation by a uvrD deletion." Genetics 141, no. 2 (October 1, 1995): 443–52. http://dx.doi.org/10.1093/genetics/141.2.443.
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Abstract Helicase II (uvrD gene product) and helicase IV (helD gene product) have been shown previously to be involved in the RecF pathway of recombination. To better understand the role of these two proteins in homologous recombination in the RecF pathway [recBCsbcB(C) background, we investigated the interactions between helD, uvrD and the following RecF pathway genes: recF, recO, recN and ruvAB. We observed synergistic interactions between uvrD ant the recF, recN, recO and recG genes in both conjugational recombination and the repair of methylmethane sulfonate (MMS)-induced DNA damage. No synergistic interactions were detected between helD and the recF, recO and regN genes when conjugational recombination was analyzed. We did, however, detect synergistic interactions between helD and recF/recO in recombinational repair. Surprisingly, the uvrD deletion completely suppressed the phenotype of a ruvB mutation in a recBCsbcB(C) background. Both conjugational recombination efficiency and MMS-damaged DNA repair proficiency returned to wild-type levels in the deltauvrDruvB9 double mutant. Suppression of the effects of the ruvB mutation by a uvrD deletion was dependent on the recG and recN genes and not dependent on the recF/O/R genes. These data are discussed in the context of two "RecF" homologous recombination pathways operating in a recBCsbcB(C) strain background.
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Wang, Xin, Carolyn A. Peterson, Huyong Zheng, Rodney S. Nairn, Randy J. Legerski, and Lei Li. "Involvement of Nucleotide Excision Repair in a Recombination-Independent and Error-Prone Pathway of DNA Interstrand Cross-Link Repair." Molecular and Cellular Biology 21, no. 3 (February 1, 2001): 713–20. http://dx.doi.org/10.1128/mcb.21.3.713-720.2001.
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ABSTRACT DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.
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Shcherbakov, Victor P. "Mismatch repair in recombination of bacteriophage T4." BioMolecular Concepts 3, no. 6 (December 1, 2012): 523–34. http://dx.doi.org/10.1515/bmc-2012-0021.
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AbstractThe review focuses on the mechanism of mismatch repair in bacteriophage T4. It was first observed in T4 as an extra recombination mechanism, which contributed to the general recombination only when particular rII mutations were used as genetic markers (high-recombination markers), whereas it was inactive toward other rII mutations (low-recombination markers). This marker-dependent recombination pathway was identified as a repair of mismatches in recombinational heteroduplexes. Comparison of the structure of markers enabled us to make several specific conclusions on the nature of the marker discrimination by the mismatch repair system operating during T4 crosses. First, heteroduplexes with one mismatched base pair (either of transition or of transversion type) as well as single-nucleotide mismatches of indel type are not efficiently repaired. Second, among the repairable mismatches, those with two or more contiguous mismatched nucleotides are the most effectively repaired, whereas insertion of one correct pair between two mismatched ones reduces the repairability. Third, heteroduplexes containing insertion mutations are repaired asymmetrically, the longer strand being preferentially removed. Fourth, the sequence environment is an important factor. Inspection of the sequences flanking mismatches shows that runs of A:T pairs directly neighboring the mismatches greatly promote repair. The mismatch is recognized by T4 endonuclease VII and nicked on the 3′ side. The nonpaired 3′ terminus is attacked by the proofreading 3′→5′ exonuclease of T4 DNA polymerase that removes the mismatched nucleotides along with several (~25) complementary nucleotides (the repair tract) and then switches to polymerization. The residual nick is ligated by DNA ligase (gp30). Most probably, the T4 system repairs replication and other mismatches as well; however, it might not discriminate old and new DNA strands and so does not seem to be aimed at repair of replication errors, in contrast to the most commonly studied examples of mismatch repair.
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Berardini, Mark, Patricia L. Foster, and Edward L. Loechler. "DNA Polymerase II (polB) Is Involved in a New DNA Repair Pathway for DNA Interstrand Cross-Links inEscherichia coli." Journal of Bacteriology 181, no. 9 (May 1, 1999): 2878–82. http://dx.doi.org/10.1128/jb.181.9.2878-2882.1999.
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ABSTRACT DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be ∼0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in apolB + (∼0.6) than in a ΔpolB(∼0.1) strain. Complementation with apolB +-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type <polB < recA < polB recA ∼ uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing,E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.
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Symington, Lorraine S. "Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair." Microbiology and Molecular Biology Reviews 66, no. 4 (December 2002): 630–70. http://dx.doi.org/10.1128/mmbr.66.4.630-670.2002.
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SUMMARY The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.
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Tamburini, Beth A., and Jessica K. Tyler. "Localized Histone Acetylation and Deacetylation Triggered by the Homologous Recombination Pathway of Double-Strand DNA Repair." Molecular and Cellular Biology 25, no. 12 (June 15, 2005): 4903–13. http://dx.doi.org/10.1128/mcb.25.12.4903-4913.2005.
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ABSTRACT Many recent studies have demonstrated recruitment of chromatin-modifying enzymes to double-strand breaks. Instead, we wanted to examine chromatin modifications during the repair of these double-strand breaks. We show that homologous recombination triggers the acetylation of N-terminal lysines on histones H3 and H4 flanking a double-strand break, followed by deacetylation of H3 and H4. Consistent with a requirement for acetylation and deacetylation during homologous recombination, Saccharomyces cerevisiae with substitutions of the acetylatable lysines of histone H4, deleted for the N-terminal tail of histone H3 or H4, deleted for the histone acetyltransferase GCN5 gene or the histone deacetylase RPD3 gene, shows inviability following induction of an HO lesion that is repaired primarily by homologous recombination. Furthermore, the histone acetyltransferases Gcn5 and Esa1 and the histone deacetylases Rpd3, Sir2, and Hst1 are recruited to the HO lesion during homologous recombinational repair. We have also observed a distinct pattern of histone deacetylation at the donor locus during homologous recombination. Our results demonstrate that dynamic changes in histone acetylation accompany homologous recombination and that the ability to modulate histone acetylation is essential for viability following homologous recombination.
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Zahradka, Ksenija, Sanela Šimić, Maja Buljubašić, Mirjana Petranović, Damir Đermić, and Davor Zahradka. "sbcB15 and ΔsbcB Mutations Activate Two Types of RecF Recombination Pathways in Escherichia coli." Journal of Bacteriology 188, no. 21 (August 25, 2006): 7562–71. http://dx.doi.org/10.1128/jb.00613-06.
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ABSTRACT Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and ΔsbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and ΔsbcB mutations on DNA repair after UV and γ irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than ΔsbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as ΔsbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC ΔsbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.
Dissertations / Theses on the topic "Recombination Repair Pathway"
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Hussain, Shobbir. "Characterisation of the fanconi anaemia pathway and its role in homologous recombination repair." Thesis, King's College London (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413559.
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Saitou, Yuuichirou. "Regulatory mechanism of damage-dependent homologous recombination." Kyoto University, 2015. http://hdl.handle.net/2433/199392.
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Kyoto University (京都大学)0048
新制・課程博士
博士(人間・環境学)
甲第19068号
人博第721号
新制||人||173(附属図書館)
26||人博||721(吉田南総合図書館)
32019
京都大学大学院人間・環境学研究科相関環境学専攻
(主査)教授 小松 賢志, 教授 宮下 英明, 准教授 三浦 智行
学位規則第4条第1項該当
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Kingham, Guy L. "Screening for inhibitors of and novel proteins within the homologous recombination DNA repair pathway." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:e2988d0b-c6d4-42a8-aef9-f320a13d6391.
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The homologous recombination (HR) pathway of DNA repair is essential for the faithful repair of double-stranded DNA breaks (DSBs) in all organisms and as such helps maintain genomic stability. Furthermore, HR is instrumental in the cellular response to exogenous DNA damaging agents such as those used in the clinic for chemo- and radiotherapy. HR in humans is a complex, incompletely understood process involving numerous stages and diverse biochemical activities. Advancing our knowledge of the HR pathway in humans aids the understanding of how chemo- and radiotherapies act and may be used to develop novel therapeutic strategies. Recent studies have identified inhibition of HR as one of the mechanisms via which a number of recently developed chemotherapeutics have their effect. Accordingly, the clinical potential of HR inhibitors is under investigation. My work has centred around the identification of both novel HR proteins and novel, small molecule HR inhibitors. To further these aims, I have successfully employed high-throughput RNAi and small molecule screening strategies. RNAi screens are commonly used to identify genes involved in a given cellular process via genetic loss of function, whilst small molecule, cell based screens are a powerful tool in the drug discovery process.
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Tay, Ye Dee. "The analysis of homologous recombination pathways in Saccharomyces cerevisiae." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:2832c80a-202d-4b92-9685-5570c25f7386.
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Homologous recombination (HR) is essential for the repair of DNA doublestrand breaks (DSBs) and damaged replication forks. However, HR can also cause gross chromosomal rearrangements (GCRs) by producing crossovers (COs), resulting in the reciprocal exchange of sequences between non-sister chromatids. Therefore, HR-mediated GCRs are suppressed via the promotion of HR pathways that favour noncrossover (NCO) formation, such as the synthesis-dependent strand annealing (SDSA) and dissolution pathways, which are modulated by Mph1 and Sgs1 helicases, respectively. The mismatch repair (MMR) pathway is intricately associated with HR via its roles in repairing mismatches on heteroduplex DNA that can arise during HR and in preventing homeologous recombination. Using a plasmid break-repair assay, we have revealed a novel, MMR-independent role of MutSα in promoting the formation of a subset of COs that is specifically supressible by Mph1, during HR between two completely homologous sequences. In contrast, the MMR-dependent function of MutSα, together with Mph1 and Sgs1, was shown to be required for the suppression of CO formation during homeologous recombination. These data indicate that Mph1 can both antagonise and promote the functions of MutSα during DSB repair, depending on the levels of homology between the two recombining sequences. COs are generated by the resolution of Holliday junction (HJ) intermediates formed at the terminal stages of HR. Several S.cerevisiae proteins such as Yen1, Mus81, Slx1 and Rad1 have been implicated in HJ resolution. However, the in vivo roles of these proteins in HJ resolution remain to be confirmed. To directly and quantitatively monitor in vivo HJ resolution in S.cerevisiae, a transformation-based HJ resolution assay using a plasmid-borne HJ substrate has been developed. Using this system, we have demonstrated an in vivo HJ resolution function of Yen1, which acts redundantly with Mus81. Moreover, these redundant activities of Yen1 and Mus81 are essential for survival during replication stress, but are dispensable for DSB repair. An Slx4 and Rad1-dependent in vivo HJ resolution activity was also observed in the absence of Yen1 and Mus81 that was suppressed by presence of Slx1. Models describing how the nucleases interact to process HJs in vivo will be discussed.
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Chu, Wai Kit. "Genetic analysis of homologous recombination repair pathways." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510942.
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Braybrooke, Jeremy P. "Characterisation of human homologues of the RAD51 protein." Thesis, Oxford Brookes University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340870.
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Cukras, Scott. "Promoting Genome Stability via Multiple DNA Repair Pathways." Scholar Commons, 2015. https://scholarcommons.usf.edu/etd/5470.
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Maintaining genome integrity is indispensible for cells to prevent and limit accruement of deleterious mutations and to promote viable cell growth and proliferation. Cells possess a myriad of mechanisms to detect, prevent and repair incurred cellular damage. Here we discuss various proteins and their accompanying cellular pathways that promote genome stability. We first investigate the NEDD8 protein and its role in promoting homologous recombination repair via multiple Cullin E3 ubiquitin ligases. We provide specific mechanisms through which, UBE2M, an E2 conjugating enzyme, neddylates various Cullin ligases to render them catalytically active to degrade their substrates by the proteasome. We show that CUL1, CUL2 and CUL4 are important in regulating various steps in the DNA damage response. Our data indicates that UBE2M and the neddylation pathway are important for genome stability. Our second topic discusses the role of the USP1- UAF1 deubiquitinating enzyme in promoting homologous recombination. We show that USP1-UAF1 interact with and stabilize RAD51AP1 (RAD51- Associated Protein 1). RAD51AP1 has previously been reported to promote homologous recombination by facilitating recombinase activity of RAD51, an essential protein involved in homologous recombination repair. We show that USP1, UAF1 and RAD51AP1 depletion leads to genome instability. Our data demonstrates the importance of these proteins in promoting genome integrity via homologous recombination.
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KATIA, CAPITANI. "Genome editing for clinically relevant mutations in genetic diseases and cancer." Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1211914.
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The present thesis concerns of two sections. The first one focuses on the application of Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR) system as a tool for precise genome targeting and genome editing; the association between specific endonuclease and RNA guides complementary to the DNA target allows its targeting with single-nucleotide precision. CRISPR/Cas is able to perform Double-Strand Breaks (DSBs) at a target site which are soon repaired by cellular repairing mechanism, non-homologous end joining (NHEJ) or homology-directed repair (HDR).
The first part of my project aims to explore and demonstrate the efficacy of a personalized therapeutic approach based on the CRISPR/Cas9 technology associated with adeno-associated viral vectors (AAVs)s, a mutation-specific gene therapy to restore mutated genes in genetic diseases to their original sequence trough the HDR-mediated correction. I developed an mCherry/EGFP reporter cassette where the reporter gene bears a mutation-specific target. It connects the mCherry and the EGFP (out of frame) coding sequences. Due to a frameshift, the reactivation of the EGFP allows the visualization of cells in which Cas9 had targeted the mutation-specific sequence leading to the production of Indels. I worked to edit mutations involved in specific genetic diseases such as mutations in FOXG1 or in MECP2, which are responsible for Rett syndrome, in the IQSEC2 gene that causes an intellectual disability clinically related to the Rett syndrome and in COL4A3 and COL4A5 causing Alport syndrome.
In the second part of my study, I worked on developing a gene editing system aims to selective targeting to cancer cells while preserving the genetic integrity of normal cells. To this aim, I plan to exploit microhomology-mediated end joining (MMEJ) through Cas12a, an RNA-directed endonuclease that causes double-strand breaks with staggered ends, to insert in-frame the Herpes Simplex Virus –Thymidine Kinase suicide gene to trigger cell death. I designed and developed a construct to target a patient-specific single nucleotide variant within a coding sequence of the TP53 gene, from a patient with Chronic Lymphocytic Leukemia characterized by clonal expansion of clones bearing this TP53 mutation. I am able to detect the proper integration of the suicide gene sequence by analyzing the treated cells by fluorescence-activated cell sorting (FACS). Indeed, a green fluorescent protein (EGFP) sequence is linked to the TK by a 2A peptide system, thus green fluorescent cells are also the one expressing for the TK gene.
The second section of my thesis concerns the COVID-19 pandemic global crisis and the need to understand how best to study and treat COVID-19. A key focus is sharing and analyzing data to learn about the genetic determinants of COVID-19 susceptibility, severity, and outcomes. In particular, my work has been focused on the TLR7 gene that has been involved as an important pattern recognition receptor for the ssRNA of SARS-CoV-2. We demonstrate that rare loss-of-function variants in the TLR7 gene in young men with severe COVID-19 and with no prior history of major chronic diseases were associated with impaired TLR7 signaling and type I and II IFN responses.
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Cataldi, Marcela Patricia. "Diverse Effects of DNA Repair Pathways on the Outcome of Recombinant Adeno-Associated Virus (rAAV) Vector Gene Delivery." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1303842573.
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Singh, Amandeep. "Exploration of the Recombination Repair Pathway in Mycobacteria : Identification and Characterization of New Proteins." Thesis, 2018. https://etd.iisc.ac.in/handle/2005/4259.
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Genomic integrity is a fundamental requisite for survival and proliferation of all organisms.
The genetic material is continuously threatened by a multitude of extrinsic and intrinsic factors. Consequently, the presence of strong DNA repair systems is essential to aid errorfree transmission of genetic material to successive generations. In prokaryotes, repair by
homologous recombination (HR) provides a major means to reinstate the genetic information lost in DNA damage. Pathogenic bacteria, such as Mycobacterium tuberculosis, face an additional threat of DNA damage due to antibiotic treatment and immune stresses inside the macrophage. Consequently, M. tuberculosis has evolved a remarkably strong DNA repair network, providing it robust survivability in the harsh environments faced inside the host cell.
The importance of HR or recombination repair in pathogenesis and emergence of antibiotic resistance in M. tuberculosis is well established. However, many aspects of the pathway remain elusive as of now. This thesis is concerned with the analysis of recombination repair system in the genus mycobacteria and characterization of two novel proteins identified in the
process.
Chapter 1 gives a detailed account of the mechanics of HR, using the well studied E. coli system and highlights the differences in mycobacteria. Recombination repair comprises a series of processes carried out by more than 20 proteins, that ultimately leads to repair of damaged DNA. Processing of DNA strands at double strand breaks and single strand gaps, to produce a 3' overhang, initiates the process. Unlike E. coli, complexes of RecFO-RecR and AdnAB-RecR provide two alternate pathways for end resection of strands and RecA loading in mycobacteria. The exchange of an undamaged strand with the damaged strand, facilitated by RecA, is central to recombination. Additionally, Single-Stranded DNA binding proteins (SSB) facilitate the loading of RecA onto the single-stranded overhang produced by the pre-processing enzymes. Resolution of strands formed due to strand exchange, via multi-stand branched DNA intermediates (such as D-loops, three-way junctions, Holliday junctions etc) by RuvABC or RecG, is the final step of recombination. An additional HJ resolvase YqgF, with unclear functions, is also present in mycobacteria. Furthermore, the major end resection enzymes (RecBCD) involved in HR in E. coli, were implicated in the Single-Strand Annealing (SSA) pathway in mycobacteria.
As part of an effort to improve the understanding of recombination repair in mycobacteria, a structure based genomic search for such proteins was carried out in 43 mycobacteria with known genome sequences (Chapter 2). Of about 20 proteins known to be involved in the pathway, a set of 9 proteins, namely, RecF, RecO, RecR, RecA, SSBa, RuvA, RuvB and RuvC was found to be indispensable among the 43 mycobacterial strains. A domain level analysis indicated that most domains involved in recombination repair are unique to these proteins and are present as single copies in the genomes. Synteny analysis reveals that the gene order of proteins involved in the pathway is not conserved, suggesting that they may be regulated differently in different species. Sequence conservation among the
same protein from different strains suggests the importance of RecO-RecA and RecFORRecA presynaptic pathways in the repair of double strand-breaks and single strand-breaks respectively. New insights into the binding of small molecules to the relevant proteins are
provided by binding pocket analysis using three-dimensional structural models. New annotations obtained from the analysis, include identification of a protein (RecGwed) with a probable Holliday junction binding role present in 41 mycobacterial genomes and that of
a RecB-like nuclease, containing a cas4 domain, present in 42 genomes. A second SingleStranded DNA Binding protein (SSBb), in addition to the canonical one (SSBa), was present in all mycobacteria except M. leprae.
Chapter 3 describes the cloning, expression, purification and structural studies on SSBb from M. smegmatis (MsSSBb). MsSSBb has been crystallized and X-ray analyzed in the first structure elucidation of a mycobacterial SSBb. The protein crystallizes in hexagonal
space group P6522 (a = b =73.61 Å, c = 216.21 Å), with half a tetrameric molecule in the asymmetric unit of the cell. In spite of the low sequence identity between SSBas and SSBbs in mycobacteria, the tertiary and quaternary structure of the DNA binding domain of MsSSBb is similar to that observed in mycobacterial SSBas. In particular, the quaternary structure is 'clamped' using a C-terminal stretch of the N-domain, which endows the tetrameric molecule with additional stability and its characteristic shape. A comparison involving available, rather limited, structural data on SSBbs from other sources, appears to suggest that SSBbs could exhibit higher structural variability than SSBas do.
It was realized that many bacterial species have a paralogous SSBb. The SSBb proteins have not been well characterized. While in B. subtilis, SSBb has been shown to be involved in genetic recombination; in S. coelicolor it mediates chromosomal segregation
during sporulation. Chapter 4 describes the distinct properties and the role of SSBb in mycobacteria. Sequence analysis of SSBs from mycobacterial species suggests low conservation of SSBb proteins, as compared to the conservation of SSBa proteins. Like most
bacterial SSB proteins, M. smegmatis SSBb (MsSSBb) forms a stable tetramer. However, solution studies indicate that MsSSBb is less stable towards thermal and chemical denaturation than MsSSBa. Also, in contrast to the 5-20 fold differences in DNA binding
affinity between paralogous SSBs observed in other organisms, MsSSBb is only about twofold poorer in its DNA binding affinity than MsSSBa. The expression levels of ssbB gene increased during UV and hypoxic stresses, while the levels of ssbA expression declined. A
direct physical interaction of MsSSBb and RecA, mediated by the C-terminal tail of MsSSBb was also established. The results obtained in this study indicate a role of MsSSBb in recombination repair during stress.
Chapter 5 describes the characterization of the previously annotated hypothetical protein RecGwed and its probable role as a novel regulator in the resolution of branched DNA structures. The protein is composed of an unusually charged N-terminus and a C-terminal
'wedge' domain, similar to the wedge domain of RecG. A database search suggested that RecGwed is predominately present in the phylum Actinobacteria, along with some other known human pathogens. Purified M. smegmatis RecGwed (MsRecGwed) exists as a stable monomer in the solution. CD studies and homology modeling indicated an unusually low content of regular secondary structures. MsRecGwed was able to bind branched DNA structures such as Holliday junction, three-way junction, three-strand junction and replication fork in vitro, while it does not interact with ss- or dsDNA. The expression of recGwed in M. smegmatis was up-regulated during stationary phase/UV damage and down-regulated during MMS/H2O2 treatment. These observations indicate the possibility of involvement of RecGwed in DNA transactions in post-replicative (stationary phase) recombination events, that proceed though branched DNA intermediates. The work described in this chapter is the first report of characterization of RecGwed-like proteins. Taken together, the work done in this thesis augments the existing repertoire of proteins known to be involved in DNA repair pathways in mycobacteria. As indicated in the concluding chapter, this study also creates a trail of future experiments that will improve our current understanding of HR in mycobacteria.
As a part of ongoing efforts in the laboratory, on the characterization of enzymes which sanitize the nucleotide pool to prevent DNA damage, structural studies on M. smegmatis MutT2 have been carried out (Appendix). Structure of the native protein, and its complexes with substrates 5me-dCTP, dCTP and CTP and the respective products, has been determined. The work presented here is the first report of MutT2-type CTP pyrophosphorylase enzymes in complex with substrates. It provides insights into the mechanism of action and the molecular basis of the functioning of mycobacterial MutT2
Book chapters on the topic "Recombination Repair Pathway"
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Gellert, Martin, and J. Fraser McBlane. "Steps along the pathway of V(D)J recombination." In DNA Repair and Recombination, 39–43. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-011-0537-8_6.
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Ishiai, Masamichi, Junya Tomida, Akiko Itaya, James Hejna, and Minoru Takata. "The Fanconi Anemia Pathway and Interstrand Cross-Link Repair." In DNA Replication, Recombination, and Repair, 175–210. Tokyo: Springer Japan, 2016. http://dx.doi.org/10.1007/978-4-431-55873-6_8.
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Takata, Minoru, Kazuhiko Yamamoto, Nobuko Matsushita, Hiroyoki Kitao, Sciki Hirano, and Masamichi Ishaiai. "The fanconi anemia pathway promotes homologous recombination repair in DT40 cell line." In Subcellular Biochemistry, 295–311. Dordrecht: Springer Netherlands, 2006. http://dx.doi.org/10.1007/978-1-4020-4896-8_17.
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Masuda, Yuji, Fumio Hanaoka, and Chikahide Masutani. "Translesion DNA Synthesis and Damage Tolerance Pathways." In DNA Replication, Recombination, and Repair, 249–304. Tokyo: Springer Japan, 2016. http://dx.doi.org/10.1007/978-4-431-55873-6_11.
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Goodenow, Donna, Kiran Lalwani, and Christine Richardson. "DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural Compounds." In DNA - Damages and Repair Mechanisms. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.95453.
Full textAbstract:
Eukaryotic cells use homologous recombination (HR), classical end-joining (C-NHEJ), and alternative end-joining (Alt-EJ) to repair DNA double-strand breaks (DSBs). Repair pathway choice is controlled by the activation and activity of pathways specific proteins in eukaryotes. Activity may be regulated by cell cycle stage, tissue type, and differentiation status. Bioflavonoids and other environmental agents such as pesticides have been shown to biochemically act as inhibitors of topoisomerase II (Top2). In cells, bioflavonoids directly lead to DNA double-strand breaks through both Top2-dependent and independent mechanisms, as well as induce DNA damage response (DDR) signaling, and promote alternative end-joining and chromosome alterations. This chapter will present differences in expression and activity of proteins in major DNA repair pathways, findings of Top2 inhibition by bioflavonoids and cellular response, discuss how these compounds trigger alternative end-joining, and conclude with implications for genome instability and human disease.
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Singh, Deepika, and Chandra Bhushan Prasad. "DNA Damage Response: A Therapeutic Landscape For Breast Cancer Treatment." In Breast Cancer: Current Trends in Molecular Research, 62–85. BENTHAM SCIENCE PUBLISHERS, 2022. http://dx.doi.org/10.2174/9781681089522112010006.
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Breast cancer is responsible for cancer-related death among women globally. The known causes of breast cancer include genetic predisposition, dysregulated hormonal signaling due to psychological stress, and aging and lifestyle factors, such as smoking and alcohol consumption. Due to improved treatment strategies, the overall survival is significantly increased; however, it is still significantly associated with death worldwide. Breast cancer's initiation and progression are strongly influenced by genomic instability. Defect in DNA damage response (DDR) pathways, which enable cells to survive, help in the accumulation of mutation, clonal selection, and expansion of cancer cells. Germline mutation in breast cancer susceptibility genes, BRCA1 and BRCA2, TP53, and PTEN, increases the risk of early onset of disease. During the initial and clonal selection of cancer cells, a defect in one DNA repair pathway could potentially be compensated by another pathway. Therefore, cancer cells with defective DNA repair pathways could be easily killed by targeting the compensatory pathways by inducing synthetic lethality. Evidently, cancer cells with defective DDR or decreased DNA repair capacity show synthetic lethality in monotherapy when the backup DNA repair pathway is inhibited. For instance, tumors with defective homologous recombination (HR) can be targeted by inhibitors of double-strand break repair enzymes. Here, we briefly addressed the relevant factors associated with the development of breast cancer and the role of the DDR factor in the development of breast cancer. In addition, recent treatment strategies targeting genomic instability in breast cancer will be summarized as well as how the genomic instability and defective DDR can be targeted for the treatment of breast cancer.
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Aqeel, Amna, Javaria Zafar, Naureen Ehsan, Qurat-Ul-Ain, Mahnoor Tariq, and Abdul Hannan. "Interstrand Crosslink Repair: New Horizons of DNA Damage Repair." In DNA - Damages and Repair Mechanisms. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.97551.
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Since the dawn of civilization, living organisms are unceasingly exposed to myriads of DNA damaging agents that can temper the ailments and negatively influence the well-being. DNA interstrand crosslinks (ICLs) are spawned by various endogenous and chemotherapeutic agents, thus posing a somber menace to genome solidity and cell endurance. However, the robust techniques of damage repair including Fanconi anemia pathway, translesion synthesis, nucleotide excision and homologous recombination repair faithfully protect the DNA by removing or tolerating damage to ensure the overall survival. Aberrations in such repair mechanisms adverse the pathophysiological states of several hereditary disorders i.e. Fanconi Anemia, xeroderma pigmentosum, cerebro-oculo-facio-skeletal syndrome and cockayne syndrome etc. Although, the recognition of ICL lesions during interphase have opened the new horizons of research in the field of genetics but still the detailed analysis of conditions in which repair should occur is largely elusive.
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"The BRCA1-BRCA2 Pathway of Homologous Recombination and the Opportunity for Synthetic Lethal Approaches to Cancer Therapy." In DNA Repair and Cancer, 488–510. CRC Press, 2013. http://dx.doi.org/10.1201/b14587-40.
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Lucchesi, John C. "DNA repair and genomic stability." In Epigenetics, Nuclear Organization & Gene Function, 173–83. Oxford University Press, 2019. http://dx.doi.org/10.1093/oso/9780198831204.003.0015.
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A number of pathways have evolved in order to repair DNA. Mismatch repair (MMR) operates when an improper nucleotide is used or when an insertion or deletion occurs during replication. Nucleotide excision repair (NER) repairs damage that distorts the DNA helix such as the presence of pyrimidine dimers induced by ultraviolet light. Base excision repair (BER) removes damaged or altered DNA bases that do not result in a conformational change in the chromatin. Single-strand break repair (SSBR) uses the same enzymatic steps as BER. Double-strand break (DSB) repair can involve either non-homologous end-joining (NHEJ) or homologous recombination (HR). In NHEJ, the broken DNA ends are joined directly. HR requires that one of the strands of the broken DNA molecule participates in the strand invasion of the sister chromatid. The site of the DSB must be modified to allow access to the repair machinery. This modification involves remodeling complexes, as well as histone-modifying enzymes.
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Beying, Natalja, Carla Schmidt, and Holger Puchta. "Double strand break (DSB) repair pathways in plants and their application in genome engineering." In Genome editing for precision crop breeding, 27–62. Burleigh Dodds Science Publishing, 2021. http://dx.doi.org/10.19103/as.2020.0082.04.
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In genome engineering, after targeted induction of double strand breaks (DSBs) researchers take advantage of the organisms’ own repair mechanisms to induce different kinds of sequence changes into the genome. Therefore, understanding of the underlying mechanisms is essential. This chapter will review in detail the two main pathways of DSB repair in plant cells, non-homologous end joining (NHEJ) and homologous recombination (HR) and sum up what we have learned over the last decades about them. We summarize the different models that have been proposed and set these into relation with the molecular outcomes of different classes of DSB repair. Moreover, we describe the factors that have been identified to be involved in these pathways. Applying this knowledge of DSB repair should help us to improve the efficiency of different types of genome engineering in plants.
Conference papers on the topic "Recombination Repair Pathway"
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Garner, Ian M., Zhuyan Su, Sawyer Hu, Yue Wu, Iain A. McNeish, Matthew J. Fuchter, and Robert Brown. "Abstract 2066: Modulation of homologous recombination repair pathway gene expression by a dual EZH2 and EHMT2 histone methyltransferase inhibitor and synergy with PARP inhibitors in ovarian cancer." In Proceedings: AACR Annual Meeting 2021; April 10-15, 2021 and May 17-21, 2021; Philadelphia, PA. American Association for Cancer Research, 2021. http://dx.doi.org/10.1158/1538-7445.am2021-2066.
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Liu, Xiaojun, Billie Nowak, Sarah Hargis, and William Plunkett. "Abstract 5667: Mechanism-based combinations of agents impacting the homologous recombination and nucleotide excision repair pathways." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-5667.
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Tripathi, Kaushlendra, Chinnadurai Mani, David Clark, Reagan Barnett, and Komaraiah Palle. "Abstract 2405: Rad18 regulates epistatic relationship between FA-BRCA and homologous recombination pathways to repair camptothecin induced DSB." In Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA. American Association for Cancer Research, 2014. http://dx.doi.org/10.1158/1538-7445.am2014-2405.
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Mazumder, Aloran, Athena Jimenez, and Svasti Haricharan. "Abstract PO-090: Coordinate dysregulation of base excision repair and homologous recombination pathways predominates in ER+/HER2- breast tumors from African American patients, and associates with worse disease-specific outcomes." In Abstracts: AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; October 2-4, 2020. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1538-7755.disp20-po-090.
Full textReports on the topic "Recombination Repair Pathway"
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Wilson, Thomas E., Avraham A. Levy, and Tzvi Tzfira. Controlling Early Stages of DNA Repair for Gene-targeting Enhancement in Plants. United States Department of Agriculture, March 2012. http://dx.doi.org/10.32747/2012.7697124.bard.
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Gene targeting (GT) is a much needed technology as a tool for plant research and for the precise engineering of crop species. Recent advances in this field have shown that the presence of a DNA double-strand break (DSB) in a genomic locus is critical for the integration of an exogenous DNA molecule introduced into this locus. This integration can occur via either non-homologous end joining (NHEJ) into the break or homologous recombination (HR) between the broken genomic DNA and the introduced vector. A bottleneck for DNA integration via HR is the machinery responsible for homology search and strand invasion. Important proteins in this pathway are Rad51, Rad52 and Rad54. We proposed to combine our respective expertise: on the US side, in the design of zincfinger nucleases (ZFNs) for the induction of DNA DSBs at any desired genomic locus and in the integration of DNA molecules via NHEJ; and on the Israeli side in the HR events, downstream of the DSB, that lead to homology search and strand invasion. We sought to test three major pathways of targeted DNA integration: (i) integration by NHEJ into DSBs induced at desired sites by specially designed ZFNs; (ii) integration into DSBs induced at desired sites combined with the use of Rad51, Rad52 and Rad54 proteins to maximize the chances for efficient and precise HR-mediated vector insertion; (iii) stimulation of HR by Rad51, Rad52 and Rad54 in the absence of DSB induction. We also proposed to study the formation of dsT-DNA molecules during the transformation of plant cells. dsT-DNA molecules are an important substrate for HR and NHEJ-mediatedGT, yet the mode of their formation from single stranded T-DNA molecules is still obscure. In addition we sought to develop a system for assembly of multi-transgene binary vectors by using ZFNs. The latter may facilitate the production of binary vectors that may be ready for genome editing in transgenic plants. ZFNs were proposed for the induction of DSBs in genomic targets, namely, the FtsH2 gene whose loss of function can easily be identified in somatic tissues as white sectors, and the Cruciferin locus whose targeting by a GFP or RFP reporter vectors can give rise to fluorescent seeds. ZFNs were also proposed for the induction of DSBs in artificial targets and for assembly of multi-gene vectors. We finally sought to address two important cell types in terms of relevance to plant transformation, namely GT of germinal (egg) cells by floral dipping, and GT in somatic cells by root and leave transformation. To be successful, we made use of novel optimized expression cassettes that enable coexpression of all of the genes of interest (ZFNs and Rad genes) in the right tissues (egg or root cells) at the right time, namely when the GT vector is delivered into the cells. Methods were proposed for investigating the complementation of T-strands to dsDNA molecules in living plant cells. During the course of this research, we (i) designed, assembled and tested, in vitro, a pair of new ZFNs capable of targeting the Cruciferin gene, (ii) produced transgenic plants which expresses for ZFN monomers for targeting of the FtsH2 gene. Expression of these enzymes is controlled by constitutive or heat shock induced promoters, (iii) produced a large population of transgenic Arabidopsis lines in which mutated mGUS gene was incorporated into different genomic locations, (iv) designed a system for egg-cell-specific expression of ZFNs and RAD genes and initiate GT experiments, (v) demonstrated that we can achieve NHEJ-mediated gene replacement in plant cells (vi) developed a system for ZFN and homing endonuclease-mediated assembly of multigene plant transformation vectors and (vii) explored the mechanism of dsTDNA formation in plant cells. This work has substantially advanced our understanding of the mechanisms of DNA integration into plants and furthered the development of important new tools for GT in plants.