Replication Stress-Induced Chromosome Breakage Is Correlated with Replication Fork Progression and Is Preceded by Single-Stranded DNA Formation

Abstract

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or “collapsed” replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.

Genome rearrangements caused by interstitial telomeric sequences in yeast

“…All these repeats stall the replication fork progression (24)(25)(26), and the (TGTGTGGG) 15 run is a particularly potent replication block. A connection between stalled replication forks and DSBs in yeast also has been observed for CTG repeats (22) and in mec1 cells treated with hydroxyurea (27). The recombinogenic repair of DSBs is known to be error prone (28), and single-stranded DNA recombination intermediates are particularly susceptible to mutations (29).…”

“…A DSB at this position likely is caused by the very potent replication block observed in strains with the (TGTGTGGG) 18 tract (24), because DSBs were observed previously at stalled replication forks in yeast (22,27). The latter likely is the result of a tight nucleoprotein complex formed at the interstitial Ytel sequences.…”

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

“…Microarray-based mapping of ssDNA has been achieved through random priming without denaturation or benzoyl naphthoyl DEAE (BND) cellulose enrichment (Buhler et al 2007). DSBs have been mapped by microarray using an end-labeling procedure (Feng et al 2011). These DNA-targeted assays are prone to in vitro artifacts and noises due to DNA denaturation and breakage.…”

“…These DNA-targeted assays are prone to in vitro artifacts and noises due to DNA denaturation and breakage. New protocols that lyse cells and label DNA in agarose plugs minimize but may not completely eliminate these problems (Feng et al 2011).…”

Def1 promotes polymerase exchange at stalled replication forks upon DNA damage

“…The strand is corrected by a DNA polymerase and religated [39]. endogenous sources such as replication of single-stranded DNA breaks, endogenously generated reactive oxygen species, and mechanical stress on the chromosomes [42,43]. DSBs differ from other types of DNA lesions in that they affect both strands of the DNA duplex and therefore prevent use of the complementary strand as a template for repair (see BER, NER, and MMR).…”