Novel role of DONSON in CMG helicase assembly during vertebrate DNA replication initiation.

CMG (Cdc45-MCM-GINS) helicase assembly at the replication origin is the culmination of eukaryotic DNA replication initiation. This process can be reconstructed in vitro using defined factors in Saccharomyces cerevisiae; however, in vertebrates, origin-dependent CMG formation has not yet been achieved partly due to the lack of a complete set of known initiator proteins. Since a microcephaly gene product, DONSON, was reported to remodel the CMG helicase under replication stress, we analyzed its role in DNA replication using a Xenopus cell-free system. We found that DONSON was essential for the replisome assembly. In vertebrates, DONSON physically interacted with GINS and Polε via its conserved N-terminal PGY and NPF motifs, and the DONSON-GINS interaction contributed to the replisome assembly. DONSON's chromatin association during replication initiation required the pre-replicative complex, TopBP1, and kinase activities of S-CDK and DDK. Both S-CDK and DDK required DONSON to trigger replication initiation. Moreover, human DONSON could substitute for the Xenopus protein in a cell-free system. These findings indicate that vertebrate DONSON is a novel initiator protein essential for CMG helicase assembly.

Mre11 exonuclease activity promotes irreversible mitotic progression under replication stress.

Mre11 is a versatile exo-/endonuclease involved in multiple aspects of DNA replication and repair, such as DSB end processing and checkpoint activation. We previously demonstrated that forced mitotic entry drives replisome disassembly at stalled replication forks in Xenopus egg extracts. Here, we examined the effects of various chemical inhibitors using this system and discovered a novel role of Mre11 exonuclease activity in promoting mitotic entry under replication stress. Mre11 activity was necessary for the initial progression of mitotic entry in the presence of stalled forks but unnecessary in the absence of stalled forks or after mitotic entry. In the absence of Mre11 activity, mitotic CDK was inactivated by Wee1/Myt1-dependent phosphorylation, causing mitotic exit. An inhibitor of Wee1/Myt1 or a nonphosphorylatable CDK1 mutant was able to partially bypass the requirement of Mre11 for mitotic entry. These results suggest that Mre11 exonuclease activity facilitates the processing of stalled replication forks upon mitotic entry, which attenuates the inhibitory pathways of mitotic CDK activation, leading to irreversible mitotic progression and replisome disassembly.

Ongoing replication forks delay the nuclear envelope breakdown upon mitotic entry.

DNA replication is a major contributor to genomic instability, and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S phase of the cell cycle. If these stalled forks persist into the M phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5'-triphosphate-induced stalled forks were able to restart with the addition of excess dCTP during early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after the NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of the NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase activities because of which DNA replication and mitosis occur in a mutually exclusive and sequential manner.

Mitotic entry drives replisome disassembly at stalled replication forks.

The disassembly of eukaryotic replisome during replication termination is mediated by CRL-dependent poly-ubiquitylation of Mcm7 and p97 segregase. The replisome also disassembles at stalled or collapsed replication forks under certain stress conditions, but the underlying mechanism is poorly understood. Here, we discovered a novel pathway driving stepwise disassembly of the replisome at stalled replication forks after forced entry into M-phase using Xenopus egg extracts. This pathway was dependent on M-CDK activity and K48- and K63-linked poly-ubiquitylation but not on CRL and p97, which is different from known pathways. Furthermore, this pathway could not disassemble converged replisomes whose Mcm7 subunit had been poly-ubiquitylated without p97. These results suggest that there is a distinctive pathway for replisome disassembly when stalled replication forks persist into M-phase.

Topoisomerase I poisoning results in PARP-mediated replication fork reversal.

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.

RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks.

In higher eukaryotes, the dynamics of replisome components during fork collapse and restart are poorly understood. Here we have reconstituted replication fork collapse and restart by inducing single-strand DNA lesions that create a double-strand break in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45-MCM-GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and polymerase ɛ onto DNA in a fashion that is dependent on RAD51 and MRE11 but independent of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process.

Studying DNA replication fork stability in Xenopus egg extract.

A crucial process to ensure cell survival and genome stability is the correct replication of the genome. DNA replication relies on complex machinery whose mechanisms are being elucidated using different model systems. A major aspect of this process, which is an intense subject of investigation, is what happens when replication forks encounter obstacles impairing their progression such as modified bases, pausing sites, and single strand breaks. The detailed biochemical analysis of DNA replication in the presence of DNA damage has been impeded by the lack of a cell-free system recapitulating DNA replication. Here we describe assays based on the vertebrate Xenopus laevis egg extract to study the biochemical aspects of replication fork stability.

Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.

The role of Rad51 in an unperturbed cell cycle has been difficult to distinguish from its DNA repair function. Here, using EM to visualize replication intermediates assembled in Xenopus laevis egg extract, we show that Rad51 is required to prevent the accumulation of single-stranded DNA (ssDNA) gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading- and lagging-strand DNA synthesis. In contrast, ssDNA gaps behind forks, which are prevalent on damaged templates, result from Mre11-dependent degradation of newly synthesized DNA strands and are suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct roles for Rad51 at replication forks, demonstrating that Rad51 protects newly synthesized DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.

The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activation of Chk1.

ATR-dependent activation of the kinase Chk1 is the initial step in signal transduction in the DNA replication checkpoint, which allows a cell to enter mitosis only after the completion of DNA replication. TopBP1-related proteins in higher eukaryotes are implicated in the replication checkpoint, but their exact role remains elusive because of their requirements for replication initiation. Here we report that the initiation function of Xenopus Cut5/TopBP1 could be entirely separated from its checkpoint function: the N-terminal half fragment, a region of Cut5 conserved through evolution, is sufficient for initiation, but is incapable of activating the checkpoint; the C-terminal half fragment, which is unique in metazoan species, is by itself capable of activating the checkpoint response without initiating replication. Upon the activation of Chk1, the Ser1131 within the C-terminal region of Cut5 is phosphorylated, and this phosphorylation is critical for the checkpoint response. Furthermore, Cut5 directly stimulated Chk1 phosphorylation in the in vitro kinase assay reconstituted with recombinant proteins and ATR immunoprecipitated from extracts. On the basis of replication protein A (RPA)-dependent loading of Cut5 on to replicating and replication-arrested chromatin, we propose that Cut5 plays a crucial role in the initial amplification step of the ATR-Chk1 signaling pathway at the stalled replication fork.

The N-terminal noncatalytic region of Xenopus RecQ4 is required for chromatin binding of DNA polymerase alpha in the initiation of DNA replication.

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 alpha and epsilon 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 alpha. 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.

Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication.

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 sufficient 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.

A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication.

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.