Double Strand Break Repair And Recombination

DNA双链断裂（DSB）可由电离辐射、烷基化损伤和复制引起，包括后链中间体处理不当。DNA断裂可能是染色体不稳定以及程序化基因修饰的一个强大来源。细胞有复杂的系统来处理dsb，包括DNA修复和检查点阻滞，以增加修复的机会。染色体中的dsb导致酵母中G2/M边界的检查点阻滞，这为修复提供了进一步的机会。dsb通过同源重组、末端连接和在断裂以外的同源区域进行单链退火来修复。几乎所有的生物体都表现出这些修复过程以及检查点阻滞。这些过程中的缺陷通常与人类疾病有关。

DNA double-strand breaks (DSB) can arise by ionizing radiation, alkylation damage, and replication, including improper processing of lagging strand intermediates. DNA breaks can be a powerful source of chromosome instability as well as programmed genetic modification. Cells have elaborate systems for dealing with DSBs, including DNA repair and checkpoint arrest to increase the opportunity for repair. DSBs in chromosomes lead to a checkpoint arrest at the G2/M boundary in yeast, which provides further opportunities for repair. DSBs are repaired through homologous recombination, end-joining , and by single-strand annealing at homologous regions beyond the breaks. Nearly all organisms exhibit these repair processes as well as checkpoint arrests. Defects in these processes are often associated with disease in humans. DNA ends must be processed to allow homologous interactions for recombination and single strand annealing. Endjoining involves only local nuclease degradation that enables interaction at microhomologies of only a few bases, The Ku and RAD50/MRE11/XRS2 (R/M/X) complexes of proteins are required for endjoining. In addition, the R/M/X complex functions in the nuclease processing of ends to provide recombination substrates. The R/M/X complex has been proposed to have a structural role that holds broken chromosomes and sister chromatids together through a Rad50/Mre11 hook-bridge structure. The Ku complex, which associates at the ends of breaks prevents excessive processing of broken ends. The balance of Ku and R/M/X can determine the extent and timing of end-processing of DSBs that in turn determines the timing of G2/M arrest adapation to a DSB. We have investigated the consequences of DSBs in various mutants and the mechanisms of handling DSBs. Our approaches have been extended to consider repair in the context of chromosomes. While DNA is the central component of chromosomes, the relationship between DNA and chromosome breaks has not been addressed nor the dynamics of DSB repair in different chromosomes. FUNCTION OF MRE11--While, the Mre11 subunit exhibits nuclease activities in vitro, the role of these activities in repair in mitotic cells has not been established. We have performed a comparative study of three mutants (Mre11-D16A, -D56N and -H125N) previously shown to have reduced nuclease activities in vitro. In ends-in and ends-out chromosome recombination assays using defined plasmid and oligonucleotide DNA substrates, mre11-D16A cells were as deficient as mre11 null strains, but defects were small in mre11-D56N and ?H125N mutants. mre11-D16A cells, but not the other mutants, also displayed strong sensitivity to ionizing radiation, with residual resistance largely dependent on the presence of the partially redundant nuclease Exo1. mre11-D16A mutants were also most sensitive to the S phase-dependent clastogens hydroxyurea and methyl methanesulfonate but, as previously observed for D56N and H125N mutants, were not defective in NHEJ. Importantly, the affinity of purified Mre11-D16A protein for Rad50 and Xrs2 was indistinguishable from wildtype and the mutant protein formed complexes with equivalent stoichiometry. Although the role of the nuclease activity has been questioned in previous studies, the comparative data suggests that the nuclease function of Mre11 is required for RMX-mediated recombinational repair and telomere stabilization in mitotic cells. DSBs IN REAL TIME -- In spite of many genetic and biochemical assays for checkpoint arrest and repair, little is known about the behavior of damaged chromosomes in the arrested cells. Furthermore, the question of the relationship between a DSB in DNA and a cytologically detectable chromosome break, as well as possible genetic controls have never been addressed. We developed a system based in the yeast Saccharomyces cerevisiae that provides for chromosome analysis in real time following the induction of a single DSB by an I-SceI endonuclease under the tight control of a GAL1promoter. We utilized tetR-CFP and LacI-GFP to mark each side of a DSB and Spc29-RFP fusion to identify the spindle poles. These proteins bind multiple repeats of their operater target sequences. This allowed us to investigate the development of a chromosome break following DNA scission and the relation to spindle pole separation and sister chromatid separation in wild type and various Ku and R/M/X mutants. We have established that the transition from DNA double-strand break measured at the molecular level to cytologically detectable chromosome break is prevented by the physical tethering function of the R/M/X complex and that the appearance of a chromosome break in vivo requires force that is transmitted through microtubules. DIFFERENTIAL DSB REPAIR IN CHROMOSOMES: CHROMOSOMAL VS DNA REPAIR. Chromosomal vs DNA repair. The 16 chromosomes of yeast vary in size with a range between ~250-2000 kb. Since the individual chromosomes can be displayed using pulse-field gel electrophoresis, we reasoned that it would be possible to address repair in individual chromosomes and genetic controls. We established that ethidium bromide staining could be used to directly assess the amount of DNA in each chromosome. As a result we were able to assess the randomness of DSB production as well as repair. Over the range of sizes from 250 to 1500 MB, induction of DSBs by ionizing radiation appears random which in logarithmically growing cells arrested in G2 was approximately 1 break/100 Gy/Mb. By comparing the reconstitution of full length chromosomes, and the patterns of breaks following exposure to various doses we are now in a position to evaluate the extent to which repair of breaks is random. This is being confirmed for a small number of chromosomes using Southern blotting approaches. There is was little if any repair of chromosomal breaks 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 type of repair during this phase of the cell cycle. The Chromosome XII-specific difference in repair is likely related to nearly half 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 required for repair between sister chromatids. 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 nearly half being repaired in 1.5 hours following a dose (800 Gy) that induces ~500 DSBs per cell. The repair requires the RAD50, RAD51 and RAD52 genes. Surprisingly, the repair is dependent on the RAD9 gene which previously was considered to be required only for checkpoint control. Using our recently developed analysis of break induction and repair in each of the chromosomes of varying sizes, we found that breaks are not simply repaired in a random fashion. Large chromosomes appear to be repaired more readily than would be predicted if the repair of DSBs is strictly random. This has implications for possible centers of repair. This is consistent with the above observation that chromosome fragments with DSBs are held together. Possibly the repair occurs in repair centers, which might correspond to foci containing repair proteins that are detected after induction of damage.