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.
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.
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 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.
•Forced entry into M-phase drives replisome disassembly at stalled replication forks.•CDK activity and K48- and K63-linked ubiquitylation is required for mitotic replisome disassembly.•Mitotic replisome disassembly is mechanistically distinct from that of replication termination.
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.
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
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.
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.
Synopsis
Vertebrate DONSON is involved in replication fork stability as a component of the replisome complex. This study reveals that DONSON also functions as an initiator protein to promote CMG (Cdc45‐MCM‐GINS) helicase assembly through the interaction with GINS in the initiation of DNA replication.
DONSON is required for CMG helicase assembly during DNA replication initiation.
DONSON physically interacts with Cdc45, GINS, and Polε via its N‐terminal disordered region.
The DONSON‐GINS interaction contributes to CMG helicase assembly.
DONSON's chromatin association during replication initiation requires the pre‐replicative complex, TopBP1, and kinase activities of S‐CDK and DDK.
S‐CDK and DDK require DONSON function to trigger replication initiation.
The microcephaly gene product DONSON emerges as a missing factor contributing to higher eukaryotic replisome formation, by interacting with GINS and DNA polymerase epsilon.
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.
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.
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