Ionizing radiation is an established source of chromosome aberrations (CAs). Although double-strand breaks (DSBs) are implicated in radiation-induced and other CAs, the underlying mechanisms are poorly understood. Here, we show that, although the vast majority of randomly induced DSBs in G 2 diploid yeast cells are repaired efficiently through homologous recombination (HR) between sister chromatids or homologous chromosomes, Ϸ2% of all DSBs give rise to CAs. Complete molecular analysis of the genome revealed that nearly all of the CAs resulted from HR between nonallelic repetitive elements, primarily Ty retrotransposons. Nonhomologous end-joining (NHEJ) accounted for few, if any, of the CAs. We conclude that only those DSBs that fall at the 3-5% 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 repeatassociated DSBs can efficiently lead to CAs and reshape the genome, they could be a rich source of evolutionary change.ectopic recombination ͉ gamma radiation ͉ genome rearrangements ͉ nonallelic homologous recombination ͉ retrotransposon

Ctp1 (aka CtIP or Sae2) collaborates with Mre11–Rad50–Nbs1 to initiate repair of DNA double strand breaks (DSBs), but its function(s) remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 harbors multivalent DNA-binding and bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal “RHR” DNA interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA bridging activity in vitro and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles for Ctp1 in binding and coordination of DSB repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CTIP-linked Seckel and Jawad syndromes.

Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.