REPAIR OF DSBS IN YEAST. Since chromosomes can be displayed as individual bands according to size using pulse-field gel electrophoresis (PFGE), it is possible to address repair of individual chromosomes and genetic controls in the budding yeast Saccharomcyes cerevisiae. There was little if any DSB repair leading to the restitution of full size chromosomal molecules in G1 diploid cells. Howeveer, repair of DSBs induced in G2 cells by IR was rapid with >90% DSBs repaired within 2 hr. The repair requires the RAD50, RAD51 and RAD52 genes. A critical early step in DSB repair and genome stability is resection of ends. While many studies with yeast have characterized resection at a unique DSB using site-specific endonucleases, we developed a novel approach for addressing resection of random, dirty-ended DSBs induced by IR. Circular chromosomes linearized by a single, random DSB migrate as a unique band during PFGE;however, within 10 min 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. Mung bean nuclease digestion revealed that the shift was due to resection and established PFGE-shift as a robust assay for detecting DSB resection. There was 1 to 2 kb resection per DSB end during repair in WT cells. In rad52 cells, which lack DSB repair, the resection rate was similar. However, in a rad50 mutant lacking the MRX complex, resection of radiation- and HO-induced DSBs was drastically reduced. Interestingly, our approach has allowed us to identify resection at one or two ends of a DSB providing unique opportunities to see early steps in recombination. We have extended the PFGE-shift approach to further dissect genetic and molecular controls of resection at random DSBs. Similar to results with a defined DSB, we found that resection is a 2-step process: a) initiation of a short, single-strand 3 tail (about 100 bases) which is determined by the MRX complex and Sae2, and b) processive 5 degradation carried out by Exo1 and Sgs1 (along with Dna2). In sae2 mutants, initiation is reduced dramatically, but this results in only a 2-fold reduction in rate of repair of IR-DSBs and only a modest reduction in survival. Loss of either EXO1 or SGS1 reduces processivity of resection about 2-fold with little effect on DSB repair or survival. However, resection length is severely reduced in an exo1 sgs1 double mutant. Yet, similar to the sae2 mutant, repair of the IR-DSBs is only decreased 2-fold and survival remains high, especially as compared to an MRX mutant. Thus, resection appears to be much greater than what is needed for efficient DSB repair and is not rate limiting in the overall repair of IR-induced DSBs, which has implications for mechanisms of DSB repair in other systems. Surprisingly, double-length linear molecules appeared in the WT and rad50 mutant within 1 hr after IR. Because the double length molecules were also found in a rad50 exo1 mutant, but not in rad52, they arise by a recombination pathway that is largely resection independent. Most studies of repair of DSBs produced by in vivo expression of endonucleases have utilized enzymes that produce cohesive-ended DSBs such as HO and EcoRI. We developed systems for expression of PvuII and EcoRV, nucleases that produce blunt end DSBs, using a rheostatable promoter with low basal activity. Expression of PvuII and EcoRV caused growth inhibition and strong cell killing in both haploid and diploid yeast cells. Surprisingly, there was little difference in sensitivities of WT cells and mutants defective in homologous recombination, nonhomologous end-joining (NHEJ), or both pathways. Although IR-induced DSBs were largely repaired within 4h, no repair of PvuII-induced breaks could be detected in diploid cells. These results indicate that, unlike DSBs with complementary single-stranded DNA overhangs, blunt-ended DSBs in yeast chromosomes are poor substrates for repair by either NHEJ or recombination. DSBS, SINGLE-STRAND BREAKS (SSBS) AND REPAIR IN HUMAN CELLS. Guided by our yeast work we developed a sensitive assay to measure DNA lesions/repair in human cells. It is based on the ability to detect changes in the circular 180 kb Epstein Barr Virus using PFGE. We have established that this is a very sensitive system for precise evaluation of DSB induction. Since any single DSB induced at random in circlar EBV will generate a unit size linear molecule, this provides a sensitive assay for addressing the components of DNA repair. We have already established conditions for addressing repair and found that following IR, approximately 50% of DSBs are repaired in 2 to 3 hr and have also demonstrated that the system can identify DNA repair inhibitors including those affecting PARP formation. . We also discovered a supercoil form of EBV that provides unique opportunities to address the induction and repair of SSBs, since a single SSB leads to relaxation of the supercoil. Thus, we are in a good position to monitor formation and repair of SSBs, DSBs as well as base damage that can be converted into measurable SSBs or DSBs in vitro, such as heat-labile sites. We anticipate that this will provide a far more sensitive and precise system for detecting various kinds of breaks than others currently available in human cells. It is now possible to address the incidence of SSBs and DSBs in the same sample under the same conditions in one PFGE lane. For example, we found that the ratio SSBs:DSBs for low dose (<10kr) IR is 6, which is consistent with previous determination by other methods. 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 yeast that provides CRB analysis following induction of a single site-specific DSB. 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 pole and sister chromatid separation. Transition from a DSB to a CRB is prevented by the physical tethering function of the MRX complex and the appearance of a CRB requires force that is transmitted through microtubules. Although there was induction of a detectable DSB, there were no cytologically detectable CRBs in WT cells, based on the lack of separation of the markers at each side of the DSB. However, 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. Surprisingly, there is a cold-sensitive component in rad50 mutants that results in nearly 40% of cells having a CRB. 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) including a mutant lacking nuclease function, 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 one 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 above). We conclude from our single molecule studies that processing of a DSB end and/or resection proteins prevent a DSB to CRB transition, possibly by making ends more accessible to tethering proteins. These findings reveal the complexity of maintaining chromosome integrity. Also, they support our view that contiguous DNA is not required to hold chromosomes together.
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