Genome-wide replication profiles of S-phase checkpoint mutants reveal fragile sites in yeast

Abstract

The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

“…However, substantial defects in replication restart appear to take place earlier than the generation of recombinatorial structures in S. cerevisiae mec1 mutants (Lopes et al 2001;Tercero and Diffley 2001;Cobb et al 2003;Tercero et al 2003;Raveendranathan et al 2006). Along these lines, it has been shown in yeast that loss of replication reinitiation coincides with the loss of replisome components from chromatin in mec1 cells (Cobb et al 2003;Raveendranathan et al 2006). These findings suggest an association between replisome disassembly and the recombinatorial processes that lead to DSB generation.…”

“…Previous studies suggest that prolonged replication stress leads to a decrease in the association of replication factors with chromatin (Cobb et al 2003;Raveendranathan et al 2006) and the elevated degradation of specific replisome components (Yoo et al 2004;Mailand et al 2006;Mamely et al 2006;Peschiaroli et al 2006;Kee et al 2009). Indeed, a more recent study has identified additional novel pathways that lead to replication factor degradation in response to replisome instability (Roseaulin et al 2013).…”

“…The generation of replication fork-associated recombination structures (e.g., reversed replication forks) and DSBs is increased in ATR/Mec1 pathway-deficient metazoan and yeast cells (Brown and Baltimore 2000;Lopes et al 2001;Myung et al 2001;Tercero and Diffley 2001;Cimprich and Cortez 2008;Branzei and Foiani 2010). However, substantial defects in replication restart appear to take place earlier than the generation of recombinatorial structures in S. cerevisiae mec1 mutants (Lopes et al 2001;Tercero and Diffley 2001;Cobb et al 2003;Tercero et al 2003;Raveendranathan et al 2006). Along these lines, it has been shown in yeast that loss of replication reinitiation coincides with the loss of replisome components from chromatin in mec1 cells (Cobb et al 2003;Raveendranathan et al 2006).…”

Genome-wide analysis of the replication program in mammals

“…However, the big challenge now is to gain mechanistic insights into the regulation of the replication program. In yeast, the new unbiased approaches have already contributed to such mechanistic questions (Alvino et al 2007;Knott et al 2009;McCune et al 2008;Raveendranathan et al 2006). Only the future will tell if similar advances will be gained in the studies of the mammalian replication program.…”

Chk1–cyclin A/Cdk1 axis regulates origin firing programs in mammals

“…However, while cis-acting origin of the replication sequence is clearly defined in prokaryotes, it is ambiguous in eukaryotes. For example, a recent whole-genome analysis using HU revealed an S phase checkpoint in budding yeast that suppresses many origins characterized as late (Feng et al 2006;Raveendranathan et al 2006). However, only a few origins fire late during S phase, and the Rad3-dependent S phase checkpoint has little effect on which origins are fired in fission yeast (Hayashi et al 2007;Heichinger et al 2006).…”