The role of Rad51 in an unperturbed cell cycle has been difficult to dissect from its DNA repair function. Here, using electron microscopy (EM) to visualize replication intermediates (RIs) assembled in Xenopus laevis egg extract we show that Rad51 is required to prevent the accumulation of ssDNA gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading and lagging strand DNA synthesis. Instead, ssDNA gaps behind forks, which are exacerbated on damaged templates, result from Mre11 dependent degradation of newly synthesized DNA strands as they can be suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct and unanticipated roles for Rad51 at replication forks demonstrating that Rad51 protects newly synthesised DNA from Mre11 dependent degradation and promotes continuous DNA synthesis.
Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics.
Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases ␣ and onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase ␣. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.
We have identified Xenopus homologs of the budding yeast Sld5 and its three interacting proteins. These form a novel complex essential for the initiation of DNA replication in Xenopus egg extracts. The complex binds to chromatin in a manner dependent on replication licensing and S-phase CDK. The chromatin binding of the complex and that of Cdc45 are mutually dependent and both bindings require Xenopus Cut5, the yeast homolog of which interacts with Sld5. On replicating chromatin the complex interacts with Cdc45 and MCM, putative components of replication machinery. Electron microscopy further reveals that the complex has a ring-like structure. These results suggest that the complex plays an essential role in the elongation stage of DNA replication as well as the initiation stage.
Fission yeast Cut5/Rad4 and its budding yeast homolog Dpb11 are required for both DNA replication and the S-phase checkpoint. Here, we have investigated the role of the Xenopus homolog of Cut5 in the initiation of DNA replication using Xenopus egg extracts. Xenopus Cut5, which shows sequence similarity to DmMus101 and HsTopBP1, is essential for DNA replication in the egg extracts. It is required for the chromatin binding of Cdc45 and DNA polymerases, but not for the formation of pre-replicative complexes or the elongation stage of DNA replication. The chromatin binding of Cut5 consists of two distinct modes. S-phase cyclin-dependent kinase (S-CDK)-independent binding is suf®cient for DNA replication while S-CDK-dependent binding is dispensable. Further, S-CDK acts after the chromatin binding of Cut5 and before the binding of Cdc45. These results demonstrate that the chromatin binding of Cut5 is required for the action of S-CDK, which in turn triggers the formation of pre-initiation complexes of DNA replication.
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