Double Strand Break Repair And Recombination

实时dsb。虽然DNA是染色体的核心组成部分，但对dsb与染色体断裂（crb）之间的关系知之甚少。我们在出芽酵母中开发了一个系统，该系统可以在诱导I-SceI内切酶诱导单个位点特异性DSB后实时提供染色体分析。我们利用ter - cfp和LacI-GFP序列结合蛋白标记DSB和Spc29-RFP融合的每一侧来识别纺锤杆，以研究DNA断裂后CRB的发展及其与纺锤杆分离和姐妹染色单体分离的关系。我们发现从DSB到细胞学上可检测的CRB的转变被MRX复合物的物理系聚功能所阻止，并且CRB的出现需要通过微管传递的力。虽然诱导了分子可检测的DSB，但在野生型细胞中没有细胞学可检测的crb，这是基于DSB两侧标记物缺乏分离。CRB仅在突变体中发现。我们发现，在DSB诱导后，MRX复合物的缺失导致15%的细胞发生CRB。crb的预防取决于Mre11复合物的结构而非核酸酶特征。CRB的产生需要通过微管传递的力。这表明另一种成分将断裂的染色体连接在一起。

DSBs IN REAL TIME. While DNA is the central component of chromosomes, there is little understanding about the relationship between DSBs and chromosome breaks (CRBs). We developed a system in budding yeast that provides chromosome analysis in real time following induction of a single site-specific DSB by an inducible I-SceI endonuclease. We utilized tetR-CFP and LacI-GFP sequence binding proteins to mark each side of a DSB and Spc29-RFP fusion to identify the spindle poles in order to investigate the development of a CRB following DNA scission and the relation to spindle pole separation and sister chromatid separation. We found that the transition from DSB to a cytologically detectable CRB is prevented by the physical tethering function of the MRX complex and that the appearance of a CRB requires force that is transmitted through microtubules. Although there was induction of a molecularly detectable DSB, there were no cytologically detectable CRBs in wild type cells, based on the lack of separation of the markers at each side of the DSB. A CRB was only revealed in mutants. We found that absence of MRX complex results in a CRB in 15% of the cells following DSB induction. The prevention of CRBs depends on the structural rather than the nuclease features of Mre11 complex. The production of a CRB requires force that is transmitted through microtubules. This suggested another component holds broken chromosomes together. We examined additional genes and conditions that might impact tethering of chromosome ends. Surprisingly, there is a cold sensitive component in rad50 mutants that results in nearly 40% of cells having a CRB. We examined the role of cohesin using a temperature-sensitive mutant mcd1-1. Following DSB induction at the restrictive temperature, there was an increase in cells with a CRB (13%). In the absence of both cohesin (mcd1-1) and MRX (rad50), 30% of cells had a CRB, demonstrating a significant role for chromosome maintenance complexes (SMC) in preventing CRBs. Since MRX is required for efficient resection of DSB ends, we also examined the role of exonuclease 1 (EXO1) in the development of CRBs. CRBs were greatly increased in an exo1 mutant (40% of cells), reaching nearly 80% for an exo1 rad50 double mutant. The actual role of resection was examined in two ways: the first identified loss of signal from of or both fluorescent markers. The second was based on our recently developed approach using pulsed field gel electrophoresis (PFGE) of chromosomal size molecules where resection of greater than 500 bp at a DSB end results in retardation of mobility (see below). Based on PFGE-shift analysis both rad50 and exo1 mutants have less resection from an induced DSB than WT. We conclude from our single molecule studies that resection proteins prevent a DSB to CRB transition, possibly by making ends more accessible to tethering proteins; the MRX complex and cohesin also may act to tether sister chromatids. These findings are important in understanding events associated with spontaneous changes in chromosomal DNA or in response to environmental agents and reveal the complexity of maintaining chromosome integrity. Also, they support our view that contiguous DNA is not required to hold chromosomes together. REPAIR OF DSBS. Since chromosomes can be displayed as individual bands according to size using PFGE, it is possible to address repair in individual chromosomes and genetic controls. Induction of DSBs by ionizing radiation is random with a yield of approximately 0.7 DSBs/10 Gy/Mb. There was little if any DSB repair leading to the restitution of full size chromosomal molecules in G1 diploid cells, except for Chromosome XII, while G2 cells exhibited efficient repair. Although a homologue was present that should allow for interchromosomal recombinational repair in G1 cells, there appears to be restrictions on this repair during this phase of the cell cycle. The Chromosome XII difference in repair is likely related due to much of this chromosome being composed of ribosomal DNA repeats. We propose that DSBs in these repeats can be repaired by a Rad51 independent, single-strand annealing mechanism rather than exchange mechanisms. These results suggest that many of the components necessary for DSB repair are present including the capability for resection. Repair of DSBs induced in G2 cells by ionizing radiation was rapid with over 90% DSBs being repaired within 2 hrs. The repair requires the RAD50, RAD51 and RAD52 genes. A critical early determinant in pathways of DSB repair and genome stability is resection of DSB ends. While many studies with budding yeast have characterized resection at a unique DSB using site-specific endonucleases, we have taken a macro genomic approach for addressing resection of random, dirty-ended DSBs induced by ionizing radiation. In G2/M cells, where opportunities for homologous DNA interactions are maximized, chromosomal restitution is first observed between 30-60 minutes after radiation. Circular chromosomes linearized by a single, random DSB migrate as a unique band during PFGE; however, within 10 min after radiation the band shifts and by 1 hr the apparent size increases 75 kb. The PFGE-shift was identical in WT, rad52 and rad51 strains but was delayed in exo1 mutants. This PFGE-shift was also seen with HO endonuclease-induced DSBs and unprotected telomere ends in a CDC13 mutant. Mung bean nuclease digestion revealed that the shift was due to resection and established PFGE-shift as a robust assay for detecting resection of random DSBs. There was 1 to 2 kb resection per DSB during repair in WT cells. In rad52 cells, which lack DSB repair, the resection rate was 2 kb/hour per DSB end. However, in a rad50 mutant lacking the MRX complex, resection of radiation- and HO-induced DSBs was drastically reduced; resection at radiation-induced DSBs was undetectable in rad50 exo1 double mutants. Thus, resection of most DSBs in G2/M cells requires the MRX complex. Growing rad50 cells exhibited somewhat more resection, but even then resection of radiation-induced DSBs was much more reduced in rad50 than in exo1mutants. Surprisingly, double-length linear molecules appeared in the WT and rad50 mutant within 1 hr after radiation. Because the double length molecules were also found In the rad50 exo1 double mutant, but not in RAD52, they arise by a recombination pathway that is largely resection independent. DSBS AND GENOME REARRANGEMENTS. Ionizing radiation is an established source of chromosome aberrations (CAs). Although DSBs are implicated, the underlying mechanisms are poorly understood. We examined individual colonies arising from cells subjected to radiation (to induce DSBs) for the appearance of CAs using Comparative Genome Hybridization and found that nearly 30-50% of survivors irradiated in G-2 have a CA. Although most randomly induced DSBs in G2 diploid yeast cells are repaired efficiently through homologous recombination (HR) between sister chromatids or homologous chromosomes, 2% of all DSBs gave rise to CAs. Molecular autopsy of the genome revealed that nearly all of the CAs resulted from HR between nonallelic repetitive elements, primarily Ty retrotransposons. We conclude that only those DSBs that fall at the 35% of the genome composed of repetitive DNA elements are efficient at generating rearrangements with dispersed small repeats across the genome, whereas DSBs in unique sequences are confined to recombinational repair between the large regions of homology contained in sister chromatids or homologous chromosomes. Because repeat-associated DSBs can efficiently lead to CAs and reshape the genome, they could be a rich source chromosomeal changes leading to disease, particularly cancer, so that Double strand breaks associated with repetitive DNA can unlock genome plasticity.