Abstract
DNA double-strand breaks (DSBs) are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs) with 3′-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS) can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3′ blocked single-strand breaks following the creation of abasic (AP) sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2)-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD) in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3′-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.
Highlights
DNA double-strand breaks (DSBs) are important sources of genome instability, giving rise to chromosomal aberrations and severe biological consequences including tumorigenesis and cell death [1,2]
We previously described a system using pulsed-field gel electrophoresis (PFGE) for analyzing in vivo repair of alkylation base damage caused by methyl methanesulfonate (MMS) [28] in yeast that is based on detection of chromosome breaks
We extend this system to a characterization of derived DSBs in G2 cells where there is the opportunity for recombinational repair between sister chromatids
Summary
DNA double-strand breaks (DSBs) are important sources of genome instability, giving rise to chromosomal aberrations and severe biological consequences including tumorigenesis and cell death [1,2]. While there is a great deal of information about direct DSBs, little is known about the contribution of single-strand lesions to the production of DSBs, single-strand lesions are generally accepted to be a source of DSBs via replication fork collapse in regions of single-strand DNA [8]. A common single-strand lesion that is generated during normal cell metabolism and repair is an apurinic/apyrimidic (AP) site, one of the most abundant DNA lesions in the cell [9,10]. As many as 10,000–200,000 single-strand lesions appear each day in mammalian cells [11,12]. Most of these are subject to base excision repair (BER), a highly coordinated process initiated by a lesionspecific glycosylase removing damaged bases and forming AP sites
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