All organisms detect DSBs and correct them efficiently. It is important to fully understand how DSBs and other types of lesions contribute to genome instability. REPAIR OF DSBS IN YEAST. Using budding yeast, we were the first to directly characterize DSBs, their repair and genetic control over 35 years ago. Recently, we modified systems for detection and revealed opportunities to address an early step in repair, resection. Since yeast chromosomes can be displayed as individual bands according to size using pulse-field gel electrophoresis (PFGE), repair of individual chromosomes can be addressed. There was little if any restitution of full size chromosomal molecules in G1 diploid cells following ionizing radiation (IR). However, DSBs induced in G2 cells were rapidly repaired: >90% DSBs within 2 hr. Repair requires RAD50, -51 and -52. A critical early step in DSB repair and genome stability is resection of ends. While many studies with yeast characterized resection at a unique DSB using site-specific endonucleases, it has been a challenge to address end events at random, dirty-ended DSBs. We developed a novel approach to address resection of IR induced DSBs. Circular chromosomes linearized by a single, random DSB migrate as a unique band during PFGE;however, within 10 min the band shifts to slower mobility and by 1 hr the apparent size increases 75 kb. This PFGE-shift was identical in WT, rad52 and rad51 strains. Mung bean nuclease digestion revealed the shift was due to resection. There was 1 to 2 kb resection per DSB end during repair in WT cells. In rad52 cells the resection rate was similar. However, in a rad50 mutant lacking the MRX complex, resection of radiation- and HO-induced DSBs was drastically reduced. As described in another project ES065073-21, we can apply the Pulse-shift approach to indirect DSBs generated during repair of closely opposed SSBs as well as single-strand gaps. Importantly, using a 2-D PFGE modification our approach has allowed us to identify for the first time resection at 1 or 2 ends of a DSB. In light of difficulties of precise assessment of events at random DSBs, we set out to examine resection at a defined DSB following expression of endonuclease I-SceI. This led to development of 2-D PFGE, which is turning out to be useful in addressing many aspects of resection. Using this approach, it is possible to determine whether the in vivo resection giving rise to an intermediate band known as m* is due to limited resection at the 2 ends of a unique DSB or just longer resection at either end. In this way we could establish the role of various repair components in coordinating resection at both ends of a DSB. Using our PFGE approaches, we provide the first system to distinguish resection at 0, 1 or both ends of DSBs. The 0- and 1-end resections predominate in MRX-null, Sae2, and Mre11 nuclease mutants, suggesting new roles for the cancer-related proteins (Ctp1 and MRN in humans) in repair, namely, efficient and coincident/coordinated resection at both ends of a DSB. We proposed that the structural features of the MRX complex are consistent with coincident/coordinated resection being due to an ability to interact with both DSB ends to directly coordinate resection. In the absence of MRX or SAE2, there is loss of coordination of end-resection. Similar to results with a defined DSB, we found resection is a 2-step process: a) initiation of a short, single-strand 3 tail (100 bases) which is determined by the MRX complex and Sae2, and b) processive 5 degradation carried out by Exo1 and Sgs1/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. Coincident resection at a clean I-SceI-induced break is much less dependent on Mre11 nuclease or Sae2, contrary to strong dependence on MRX complex. These results suggest a differential role for these functions at dirty and clean DSB ends. The observed MRX coordination of end-resection fits very nicely with our earlier finding of a role for MRX in physically holding the ends of a chromosome break in close proximity. 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 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 overall repair of IR-induced DSBs. Possibly its most important role is providing large ssDNA regions for signaling to prevent cell progression. TELOMERE PROCESSING. Telomere ends of chromosomes can appear as DSBs including 3 protruding ends. Telomere specific proteins can protect the ends from resection, and there have been many studies on resection after telomere uncapping in yeast and human cells. In budding yeast, when telomeres become uncapped in a cdc13-1 mutant at the restrictive temperature, the amount of resection can be large and was important in studying mutagenesis in ssDNA , while in humans the extent of resection is much smaller (<1 kb). Our approach to addressing 1- and 2-end resections and the ability to quantitate length of resection with nucleases provides a useful tool to understanding components that determine resection. We initiated studies to investigate telomere resection on individual chromosomes, multiple chromosomes and the role of nucleases. Using our PFGE shift approach, we are able to address the roles for endcapping protein Cdc13 and Ku in preventing resection of one or both ends of a chromosome, similar to what was described above for a DSB in a circle. This provides unique opportunities to address differences in stability of telomere ends among the chromosomes, the roles of various components in endprocessing (described above) such as Mre11 and Sae2 (Ctp1) and lengths of resection, when combined with in vitro nuclease treatment. We anticipate being able to integrate commonalities in processing of very different kinds of DSB ends in the same cells: telomeres, clean and dirty DSBs. GENERALIZING PFGE-SHIFT TO ADDRESS RESECTION IN OTHER ORGANISMS. Prior to our PFGE-shift approach, there were no systems that provided a ready means for rapid quantitation of resection at random DSBs across the genome, although there were several qualitative foci-based approaches (such as RPA or BRDU) to identify resection in a variety of cell types, including human. Given the large impact of relatively small single-strand tails (i.e., a few 100 bases or more), we have begun to develop a 2-D approach that captures the PFGE-shifting effect of ssDNA tails to identify and quantitate resected molecules across the genome in other organisms without using a circular chromosome. This would provide opportunities to examine components of endprocessing as well as pathway decision-making via resection such as end-joining or recombinational repair. This approach is being applied to fission yeast and eventually to human cells. 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 based on the ability to detect changes in the circular 180 kb Epstein Barr Virus using PFGE. Following IR, 50% of DSBs are repaired in 2-3 hr. We also discovered EBV supercoils, providing opportunities to address induction and repair of SSBs, since a single SSB relaxes the supercoil. We now have a far more sensitive and precise system for detecting various kinds of breaks than others currently available in human cells as well as opportunities to address SSBs and DSBs in the same sample in one PFGE lane.

Project Start
Project End
Budget Start
Budget End
Support Year
32
Fiscal Year
2013
Total Cost
$401,972
Indirect Cost
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McKinney, Jennifer Summers; Sethi, Sunaina; Tripp, Jennifer DeMars et al. (2013) A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae. BMC Genomics 14:251
Nakai, Wataru; Westmoreland, Jim; Yeh, Elaine et al. (2011) Chromosome integrity at a double-strand break requires exonuclease 1 and MRX. DNA Repair (Amst) 10:102-10
Ajiro, Kozo; Bortner, Carl D; Westmoreland, Jim et al. (2008) An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: antagonism by physiological concentrations of K(+) ions. Exp Cell Res 314:1237-49
Hazkani-Covo, Einat; Covo, Shay (2008) Numt-mediated double-strand break repair mitigates deletions during primate genome evolution. PLoS Genet 4:e1000237