REPAIR OF DSBS IN YEAST. 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 0.6 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 repair in G1. The Chr XII difference in repair is likely related 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 >90% DSBs repaired within 2 hrs. 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 budding yeast have characterized resection at a unique DSB using site-specific endonucleases, we took a macro genomic approach for addressing resection of random, dirty-ended DSBs induced by gamma radiation. In G2/M cells, where opportunities for homologous DNA interactions are maximized, chromosomal restitution is first observed between 30-60 min after radiation. 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. 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 DSBs. 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;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. Log phase rad50 cells exhibited somewhat more resection, but even then resection was much more reduced in rad50 than in exo1 mutants. 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. 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. These findings have now been extended to additional mutants, including SAE2. DSBS, SINGLE-STRAND BREAKS (SSBS) AND REPAIR IN HUMAN CELLS. Guided by our yeast work we developed a sensitive assay to measure various DNA lesions and their repair in human cells. It is based on the ability to detect changes in the circular 180 kb Epstein Barr Virus using PFGE. This virus is commonly used in generation of immortalized lymphoblast cell lines and is naturally present in latent (nonintegrated form) infections in over half the human population. We have established that this is a very sensitive system for precise evaluation of DSB induction. 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 unique 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 run of samples with PFGE. For example, we found that the ratio SSBs:DSBs for low dose (<10kr) ionizing radiation is 6, which is consistent with previous determination by other methods. 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 ionizing radiation, approximately 50% of DSBs are repaired in 2 to 3 hours and have also demonstrated that the system can be used to identify DNA repair inhibitors. 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 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 cytologically detectable 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 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. 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%). 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 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 above). 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 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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