Multiple Mechanisms Control Chromosome Integrity after Replication Fork Uncoupling and Restart at Irreparable UV Lesions

Abstract

DNA replication forks pause in front of lesions on the template, eventually leading to cytotoxic chromosomal rearrangements. The in vivo structure of damaged eukaryotic replication intermediates has been so far elusive. Combining electron microscopy (EM) and two-dimensional (2D) gel electrophoresis, we found that UV-irradiated S. cerevisiae cells uncouple leading and lagging strand replication at irreparable UV lesions, thus generating long ssDNA regions on one side of the fork. Furthermore, small ssDNA gaps accumulate along replicated duplexes, likely resulting from repriming events downstream of the lesions on both leading and lagging strands. Translesion synthesis and homologous recombination counteract gap accumulation, without affecting fork progression. The DNA damage checkpoint contributes to gap repair and maintains a replication-competent fork structure. We propose that the coordinated action of checkpoint, recombination, and translesion synthesis-mediated processes at the fork and behind the fork preserves the integrity of replicating chromosomes by allowing efficient replication restart and filling the resulting ssDNA gaps.

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…The result is a lagging strand with a gap comprising the lesion (Svoboda and Vos, 1995) to be repaired at a later time, after gap filling, by either TLS or HR. Although a similar mechanism has been proposed, when the leading strand polymerase stops at a lesion on the template (Heller and Marians, 2006;Lopes et al, 2006), an alternative possibility is decoupling of the leading and lagging strand polymerase with the lagging strand being further elongated for another 1 or 2 kb (Cordeiro-Stone et al, 1999;Pagés and Fuchs, 2003). The lagging strand could provide a template for elongation of the terminated leading strand.…”

“…Tolerance of replication blocking lesions requires alternative mechanisms, such as translesion DNA synthesis (TLS), a generally deemed error-prone bypass process and fork regression or homologous recombination (HR), allowing error-free bypass of replication-blocking lesions (Ulrich, 2005;Li and Heyer, 2008). Recently, also repriming on the leading strand has been discussed as a potential errorfree mechanism (Lopes et al, 2006).…”

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…Presumably, as in E. coli, processing of the "chicken foot" structure of the reversed fork by endonucleases such as Mus81 may generate a DSB [37]; alternatively, the stalled fork might be attacked directly. A recent study by Lopes et al in UV-irradiated NER-deficient rad14 mutants of S. cerevisiae revealed the existence of DSGs in both leading and lagging strands downstream of the replication fork [38]. This suggests that S. cerevisiae may possess a recombination-independent mechanism of leading strand replication restart, since recombination-mediated restart need not generate leading strand DSGs [34].…”

“…If replication restart on both strands can be accomplished without recombination, the problem of DSG repair might be addressed subsequent to the passage of the replication complex, allowing scheduled DNA synthesis to progress independently of the slower DSG repair processes [38]. Lopes et al examined the effect of inactivating TLS or HR functions on the distribution of DSGs in UV irradiated rad14 mutants, and found that gaps nearer the fork accumulate in TLS mutants, whereas those distant from the fork accumulate more prominently in HR mutants.…”

“…Despite this, HR seems to play a quantitatively greater role than TLS in DSG repair, suggesting that TLS often fails to bypass the lesion successfully. Interestingly, resolution of DSGs closer to the fork is also impaired in rad53 checkpoint mutants, raising the possibility that DNA damage checkpoint signaling regulates engagement of TLS polymerases [38].…”