Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.

Gene amplification plays important roles in the progression of cancer and contributes to acquired drug resistance during treatment. Amplification can initiate via dicentric palindromic chromosome production and subsequent breakage-fusion-bridge cycles. Here we show that, in fission yeast, acentric and dicentric palindromic chromosomes form by homologous recombination protein-dependent fusion of nearby inverted repeats, and that these fusions occur frequently when replication forks arrest within the inverted repeats. Genetic and molecular analyses suggest that these acentric and dicentric palindromic chromosomes arise not by previously described mechanisms, but by a replication template exchange mechanism that does not involve a DNA double-strand break. We thus propose an alternative mechanism for the generation of palindromic chromosomes dependent on replication fork arrest at closely spaced inverted repeats.[Keywords: RTS7; template switch; recombination; gene amplification; breakage-fusion-bridge] Supplemental material is available at http://www.genesdev.org.

In response to DNA damage and replication pausing, eukaryotes activate checkpoint pathways that prevent genomic instability by coordinating cell cycle progression with DNA repair. The intra-S-phase checkpoint has been proposed to protect stalled replication forks from pathological rearrangements that could result from unscheduled recombination. On the other hand, recombination may be needed to cope with either stalled forks or double-strand breaks resulting from hydroxyurea treatment. We have exploited fission yeast to elucidate the relationship between replication fork stalling, loading of replication and recombination proteins onto DNA, and the intra-S checkpoint. Here, we show that a functional recombination machinery is not essential for recovery from replication fork arrest and instead can lead to nonfunctional fork structures. We find that Rad22-containing foci are rare in S-phase cells, but peak in G2 phase cells after a perturbed S phase. Importantly, we find that the intra-S checkpoint is necessary to avoid aberrant strand-exchange events during a hydroxyurea block.

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Although the molecular enzymology of DNA replication is well characterised, how and why it occurs in discrete nuclear foci is unclear. Using fission yeast, we show that replication takes place in a limited number of replication foci, whose distribution changes with progression through S phase. These sites define replication factories which contain on average 14 replication forks. We show for the first time that entire foci are mobile, able both to fuse and re-segregate. These foci form distinguishable patterns during S phase, whose succession is reproducible, defining early-, mid-and late-S phase. In wild-type cells, this same temporal sequence can be detected in the presence of hydroxyurea (HU), despite the reduced rate of replication. In cells lacking the intra-S checkpoint kinase Cds1, replication factories dismantle on HU. Intriguingly, even in the absence of DNA damage, the replication foci in cds1 cells assume a novel distribution that is not present in wild-type cells, arguing that Cds1 kinase activity contributes to the spatio-temporal organisation of replication during normal cell growth.

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase ␦ has not been clearly defined. We show that the third subunit of human DNA polymerase ␦, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase ␦ activity and for binding of the cell cycle inhibitor p21Cip1 . In accordance with this, a peptide containing the PCNA-binding domain of p21Cip1 inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase ␦. Furthermore, pull-down assays showed that DNA polymerase ␦ requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase ␦ displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21 Cip1 . Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase ␦ within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase ␦.DNA replication requires the recruitment of multiple components during the S phase of the cell cycle. Of chief importance are DNA polymerases, of which three, ␣, ␦, and ⑀, have been shown by genetic studies in the yeast Saccharomyces cerevisiae to be essential for the replication of the eucaryotic genome (1, 2). Among these, DNA polymerase ␣-primase is the only enzyme that can start DNA synthesis de novo. It is thus ideally suited for the synthesis of short primers that serve to initiate leading strand synthesis at the replication origin and Okazaki fragments on the lagging strand (3, 4). Both DNA polymerases ␦ and ⑀ can elongate primers synthesized by DNA polymerase ␣ and appear to have overlapping or complementary functions (5). Although DNA polymerase ␦ alone can replicate both leading and lagging strands in vitro (5), mounting evidence suggests that DNA polymerase ⑀ may be implicated in DNA replication in ways that may not necessarily involve its DNA polymerase activity (6 -10).We have been interested in how DNA polymerase ␦ interacts with other factors to accomplish replication of the mammalian genome. Extensive research over a number of years has shown that DNA polymerase ␦ requires interactions with at least two factors, a toroidal-shaped homotrimeric protein known as proliferating cell nuclear antigen (PCNA) 1 and a heteropentameric complex termed replication factor C. In the presence of ATP, replication factor C interacts with the 3Ј primer-template terminus and recruits PCNA onto the DNA, creating a mobile platform that tethers DNA polymerase ␦ to the primer terminus. Once bound to PCNA, DNA polymerase ␦ acquires new dynamic properties, including high processivity, low nonpro...

A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H2O2, GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex.

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.