Добірка наукової літератури з теми "DNA Double-strand Break Repair (DSRB) Pathway"

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.

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.

Replication timing (RT) is a cellular program to coordinate initiation of DNA replication in all origins within the genome. RIF1 (replication timing regulatory factor 1) is a master regulator of RT in human cells. This role of RIF1 is associated with binding G4-quadruplexes and changes in 3D chromatin that may suppress origin activation over a long distance. Many effects of RIF1 in fork reactivation and DNA double-strand (DSB) repair (DSBR) are underlined by its interaction with TP53BP1 (tumor protein p53 binding protein). In G1, RIF1 acts antagonistically to BRCA1 (BRCA1 DNA repair associated), suppressing end resection and homologous recombination repair (HRR) and promoting non-homologous end joining (NHEJ), contributing to DSBR pathway choice. RIF1 is an important element of intra-S-checkpoints to recover damaged replication fork with the involvement of HRR. High-resolution microscopic studies show that RIF1 cooperates with TP53BP1 to preserve 3D structure and epigenetic markers of genomic loci disrupted by DSBs. Apart from TP53BP1, RIF1 interact with many other proteins, including proteins involved in DNA damage response, cell cycle regulation, and chromatin remodeling. As impaired RT, DSBR and fork reactivation are associated with genomic instability, a hallmark of malignant transformation, RIF1 has a diagnostic, prognostic, and therapeutic potential in cancer. Further studies may reveal other aspects of common regulation of RT, DSBR, and fork reactivation by RIF1.

ABSTRACT The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.

Abstract The aim of this work was to investigate the radiosensitization effects of genistein on mice sarcoma cells and the corresponding biological mechanisms in vitro and in vivo . Using the non-toxic dosage of 10 μM genistein, the sensitizer enhancement ratios after exposure to X-rays at 50% cell survival (IC 50 ) was 1.45 for S180 cells. For mice cotreated with genistein and X-rays, the excised tumor tissues had reduced blood vessels and decreased size and volume compared with the control and irradiation-only groups. Moreover, a significant increase in apoptosis was accompanied by upregulation of Bax and downregulation of Bcl-2 in the mitochondria, and lots of cytochrome c being transferred to the cytoplasm. Furthermore, X-rays combined with genistein inhibited the activity of DNA-PKcs, so DNA-injured sites were dominated by Ku70/80, leading to incompleteness of homologous recombination (HR) and non-homologous end-joining (NHEJ) repairs and the eventual occurrence of cell apoptosis. Our study, for the first time, demonstrated that genistein sensitized sarcoma cells to X-rays and that this radiosensitizing effect depended on induction of the mitochondrial apoptosis pathway and inhibition of the double-strand break (DSB) repair pathways.

The process of information exchange between two homologous DNA duplexes is known as homologous recombination (HR) or double-strand break repair (DSBR), depending on the context. HR is the fundamental process underlying the genome shuffling that expands genetic diversity (for example during meiosis in eukaryotes). DSBR is an essential repair pathway in all three domains of life, and plays a major role in the rescue of stalled or collapsed replication forks, a phenomenon known as recombination-dependent replication (RDR). The process of HR in the archaea is gradually being elucidated, initially from structural and biochemical studies, but increasingly using new genetic systems. The present review focuses on our current understanding of the structures, functions and interactions of archaeal HR proteins, with an emphasis on recent advances. There are still many unknown aspects of archaeal HR, most notably the mechanism of branch migration of Holliday junctions, which is also an open question in eukarya.

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G2-phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

10514 Background: Alterations in the DNA double-strand break repair (DSBR) pathway are associated with cancer risk. Mutations in the genes BRCA1/2 disrupt DNA DSBR. Variations in breast cancer penetrance among BRCA1/2 mutation carriers, and familial patterns among women without known BRCA1/2 mutations may be related to polymorphisms of genes in the DNA-DSBR pathway. Methods: Using a case-control study design with individuals in the UCLA Family Cancer Registry (FCR), we examined the independent effects of 100 SNPs in 19 DNA-DSBR genes. SNPs were assayed using the Applied Biosystems SNPlex™ assay. Results: 630 consecutive females from 508 families in the UCLA FCR selected for familial risk of breast cancer were included in the study. Table 1 describes select subject characteristics. Preliminary association analysis of the Caucasian subset using a nonparametric permutation method, which controls for Ashkenazi Jewish heritage and dependencies among relatives, suggests that polymorphisms within RAD21 (p=0.0044, p=0.0005), XRCC2 (p = 0.0069), XRCC4 (p =0.0511), and BRIP1 (p =0.0107) may be associated with a change in risk of breast cancer. Using only unrelated Caucasian subjects in a logistic regression analysis with covariates such as age, BMI, and Ashkenazi Jewish heritage, the effects of these polymorphisms remain significant, with 32% to 74% change in the odds of breast cancer. Conclusions: We have identified five potential SNPs in genes in the DNA-DSBR pathway that appear to be associated with a change in risk of breast cancer. This hypothesis generating study lends support to a role for polymorphisms of the DNA-repair pathway in breast carcinogenesis. Assessment of gene-environment and gene-gene interactions will help to elucidate carcinogenic mechanisms. Further validation in similar populations is warranted. (Funded by the Breast Cancer Research Foundation and NIH/NCI CA87949 R25 Career Development Program.) [Table: see text] No significant financial relationships to disclose.

The PI3K/Akt/mTOR pathway is frequently altered in human papillomavirus (HPV)-positive and negative squamous cell carcinoma of the head and neck (HNSCC) and overstimulation is associated with poor prognosis. PI3K drives Akt activation and constitutive signaling acts pro-proliferative, supports cell survival, DNA repair, and contributes to radioresistance. Since the small molecule NVP-BEZ235 (BEZ235) is a potent dual inhibitor of this pathway, we were interested whether BEZ235 could be an efficient radiosensitizer. The 50 nM BEZ235 was found to abrogate endogenous and irradiation-induced phosphorylation of Akt (Ser473). The anti-proliferative capacity of the drug resulted in an increase in G1-phase cells. Repair of radiation-induced DNA double-strand breaks (DSBs) was strongly suppressed. Reduction in DSB repair was only apparent in G1- but not in G2-phase cells, suggesting that BEZ235 primarily affects non-homologous end joining. This finding was confirmed using a DSB repair reporter gene assay and could be attributed to an impaired phosphorylation of DNA-PKcs (S2056). Cellular radiosensitivity increased strongly after BEZ235 addition in all HNSCC cell lines used, especially when irradiated in the G0 or G1 phase. Our data indicate that targeting the PI3K/Akt/mTOR pathway by BEZ235 with concurrent radiotherapy may be considered an effective strategy for the treatment of HNSCC, regardless of the HPV and Akt status.

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

To counteract the potentially calamitous effects of genomic instability in the form of double-strand breaks (DSBs), cells have evolved with two major mechanisms. First, DNA non¬homologous end joining (NHEJ) which requires no significant homology, and second, homologous recombination (HR) that uses intact sequences on the sister chromatid or homologous chromosome as a template to repair the broken DNA. Although NHEJ repairs DSBs in all stages of cell cycle; it is generally error-prone due to insertions or deletions of few nucleotides at the breakpoint. In contrast, DSBs that are generated during S and G2 phase of the cell are preferentially repaired by HR that utilizes neighboring sister chromatid as a template. A central role in the HR reaction is promoted by the RAD51 recombinase which polymerizes onto single-stranded DNA (ssDNA), catalyzes pairing and strand invasion with homologous DNA molecule. Assembly of RAD51 monomers onto ssDNA is a relatively slow process and is facilitated by several mediator proteins. The tumor suppressor protein BRCA2 is the best-characterized RAD51 mediator in DSB repair by HR. Many reports in the past two decades have established that RAD51 recruitment at break sites also depends on the RAD51 paralogs. Mammalian cells encode five RAD51 paralogs; RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 which share 20–30% identity at amino acid level with RAD51 and with each other. In addition to their role in HR, RAD51 paralogs have been identified to be involved in DNA damage signaling and replication fork maintenance. In addition, mouse knockout of RAD51 paralogs causes early embryonic lethality. Recent studies show that germline mutations in all five RAD51 paralogs cause various types of cancer including breast and ovarian cancers. Pedigree analyses revealed that similar to BRCA1 and BRCA2, pathological missense mutants of RAD51C were of high penetrance. Historically, defects in the DNA repair pathways have been exploited for cancer chemo-and radiotherapy. In an attempt to develop better cancer therapeutic approaches, the concept of synthetic lethality for cancer therapy has been recently proposed. One such example is the use of PARP1 inhibitors to treat tumors carrying mutations in HR genes, such as BRCA1 and BRCA2. Inhibition of PARP1 compromises single-strand break repair (SSBR) pathway. Upon replication fork collision, the accumulated SSBs are converted to one-ended DSBs, which are efficiently repaired by the HR for cell survival. As a result, HR-deficient tumors with BRCA1-or BRCA2-deficiency exhibit extreme sensitivity to PARP-1 inhibition resulting in cell death. This approach was highly successful in targeting tumors with severe defects in Fanconi anemia (FA)-BRCA proteins which led to PARP inhibitors being tested in clinical trials. However, targeting cancer cells that express hypomorphic mutants of HR proteins is highly challenging since such partially functional mutants require a high dosage of PARP inhibitors for effective sensitization which renders normal cells toxic and can also lead to tumor resistance. The pathological RAD51C mutants that were identified in breast and ovarian cancer patients are hypomorphic with partial repair function. The first part of my Ph.D. thesis is aimed at developing an effective strategy to target cells that express hypomorphic RAD51C mutants. To this end, we used RAD51C deficient CL-V4B hamster cells and expressed the pathological RAD51C mutants associated with breast and ovarian cancers. Cells expressing RAD51C mutants that were severely defective for HR function exhibited high sensitivity to low doses of PARP1 inhibitor (4-ANI). These cells also accumulated in G2/M and displayed chromosomal aberrations. However, RAD51C mutants that were hypomorphic were partially sensitized even at higher concentrations of PARP inhibitor. RAD51C/ CL-V4B cells displayed higher PARP activity compared WT V79B cells. Notably, PARP activity was directly proportional to the sensitivity of RAD51C mutants towards 4-ANI where highly sensitive RAD51C mutants showed higher PARP activity and vice versa. Increased PARP activity was associated with replication stress as confirmed by an increase of PARP activity in cells treated with replication stress inducer, hydroxyurea (HU). Notably, treatment of CL-V4B cells with PARP1 inhibitor (4-ANI) resulted in the accumulation of PARP1 onto the chromatin which eventually led to the formation of DSBs which suggests that PARP1 entrapment triggers replication fork collapse leading to one-ended DSBs in S-phase. To further understand the molecular mechanism of PARP inhibitor-induced toxicity of RAD51C deficient cells, we carried out chromatin fractionation from V79B and CL-V4B cells at varying time points of 4-ANI treatment. Surprisingly, there was an enhanced loading of NHEJ proteins on chromatin in CL-V4B compared to V79B cells. Consistently, an increased error-prone NHEJ was observed in CL-V4B cells which resulted in increased chromosomal aberrations and cell death. Furthermore, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV restored this phenotype. Thus, error-prone NHEJ in collaboration with PARP inhibition sensitizes RAD51C deficient cells. Since ionizing radiation (IR) is known to stimulate NHEJ activity, we hypothesized that irradiation in combination with PARP inhibitor would further sensitize the RAD51C deficient tumors. Strikingly, stimulation of NHEJ by a low dose of IR in the PARP inhibitor-treated RAD51C deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity ‘synergistically’. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a ‘synergistic approach’ and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other HR pathway genes. In addition to nuclear functions, RAD51 paralogs RAD51C and XRCC3 have been shown to localize to mitochondria and contribute to mitochondrial genome stability. However, the molecular mechanism by which RAD51 and RAD51 paralogs carry out this function is unclear. The second part of my thesis was dedicated to studying whether RAD51C/XRCC3 facilitates mitochondrial DNA replication and the underlying mechanism by which RAD51C/XRCC3 participate in mitochondrial genome maintenance during unperturbed conditions. Using mitochondrial subfractionation we show that RAD51 and RAD51 paralogs (RAD51C and XRCC3) are an integral part of mitochondrial nucleoid and absence of RAD51C/XRCC3 and RAD51 prevents the restoration of mtDNA upon depletion of mtDNA. This suggests that RAD51 and RAD5C/XRCC3 participate in mtDNA replication. To determine whether this function of RAD51C is exclusive to mitochondria we expressed NLS mutant of RAD51C which was defective for nuclear functions. Interestingly, cells expressing RAD51C R366Q were able to efficiently repopulate the depleted mtDNA after EtBr stress similar to that of WT RAD51C expressing cells, suggesting a nuclear independent function of RAD51C in mitochondrial genome maintenance. mtDNA-IP analysis revealed that RAD51 and RAD51C/XRCC3 are recruited to the mtDNA control regions spontaneously along with mitochondrial polymerase POLG. Moreover, RAD51 was found to associate with TWINKLE helicase and this association was required for the recruitment of RAD51 and RAD51C/XRCC3 at the D-loop. As in nucleus, mtDNA replisome also encounters replication stresses like altered dNTP pools, a collision between replication and transcription machinery, rNTP incorporation, oxidative stress which hampers replication fork progression. Using Dideoxycytidine (ddC) as replication stress inducer in mitochondria, we observed nearly 3-4 fold enrichment of RAD51, RAD51C, XRCC3 and POLG at the mtDNA mutation hotspot region D310. Notably, RAD51C/XRCC3 deficient cells exhibited increased lesions in the mitochondrial genome spontaneously, pointing towards the importance of RAD51C/XRCC3 in the prevention of mtDNA lesions. Moreover, RAD51C/XRCC3 deficiency prevented the repair of ddC induced mtDNA lesions. Given that RAD51C/XRCC3 and RAD51 are localized to mtDNA control regions along with POLG and their deficiency affects mtDNA replication we were curious to learn the effect of RAD51C/XRCC3 deficiency on the recruitment of POLG in mtDNA. To test this we performed a mtDNA-IP assay of POLG in RAD51C deficient cells which revealed that deficiency of RAD51C/XRCC3 and RAD51 affected the recruitment of POLG on mtDNA control regions. As a consequence RAD51C/XRCC3 deficient cells exhibit aberrant mitochondrial functions. These findings propose a mechanism for a direct role of RAD51C/XRCC3 in maintaining the mtDNA integrity under replication stress conditions.

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.

Radiation is one of the causative agents for the induction of DNA damage in biological systems. There is various possibility of radiation exposure that might be natural, man-made, intentional, or non-intentional. Published literature indicates that radiation mediated cell death is primarily due to DNA damage that could be a single-strand break, double-strand breaks, base modification, DNA protein cross-links. The double-strand breaks are lethal damage due to the breakage of both strands of DNA. Mammalian cells are equipped with strong DNA repair pathways that cover all types of DNA damage. One of the predominant pathways that operate DNA repair is a non-homologous end-joining pathway (NHEJ) that has various integrated molecules that sense, detect, mediate, and repair the double-strand breaks. Even after a well-coordinated mechanism, there is a strong possibility of mutation due to the flexible nature in joining the DNA strands. There are alternatives to NHEJ pathways that can repair DNA damage. These pathways are alternative NHEJ pathways and single-strand annealing pathways that also displayed a role in DNA repair. These pathways are not studied extensively, and many reports are showing the relevance of these pathways in human diseases. The chapter will very briefly cover the radiation, DNA repair, and Alternative repair pathways in the mammalian system. The chapter will help the readers to understand the basic and applied knowledge of radiation mediated DNA damage and its repair in the context of extensively studied NHEJ pathways and unexplored alternative NHEJ pathways.

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.

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.