DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

Studies on plasmid replication. V Replicative intermediates.

A procedure has been developed for the study of rapidly labeled intermediates in plasmid replication in normally growing bacteria. Pulse-labeled cells are enzymatically lysed on top of a neutral sucrose gradient and centrifuged so that the chromosomal DNA forms a pellet and the plasmids (and other smaller DNA molecules) form bands in the gradient. Analysis of penicillinase plasmid replication in Staphylococcus aureus has revealed that although pulse-labeled intermediates sediment faster than the 60 S circular duplex monomeric plasmid molecules, they do not have stable superhelical structure. The conversion of partially polymerized molecules, having a sedimentation coefficient of about 58 S, to fully polymerized terminal forms appears to involve a progressive change in sedimentation rate from 58 S to 67 S. Conversion of the presumably dimeric terminal forms to mature closed circular monomers is a slow and rate-limiting multi-step process (taking some 3 to 4 min at 37 °C).     

When replication forks stop

DNA synthesis is an accurate and very processive phenomenon, yet chromosome replication does not proceed at a constant rate and progression of the replication fork can be impeded. Several structural and functional features of the template can modulate the rate of progress of the replication fork. These include DNA secondary structures, DNA damage and occupied protein-binding sites. In addition, prokaryotes contain sites where replication is specifically arrested. DNA regions at which the replication machinery is blocked or transiently slowed could be particularly susceptible to genome rearrangements. Illegitimate recombination, a ubiquitous phenomenon which may have dramatic consequences, occurs by a variety of mechanisms. The observation that some rearrangements might be facilitated by a pause in replication could provide a clue in elucidating these processes. In support of this, some homologous and illegitimate recombination events have already been correlated with replication pauses or arrest sites.     

De novo assembly of genuine replication forks on an immobilized circular plasmid in Xenopus egg extracts

We describe an improved model of DNA replication in Xenopus egg extracts, in which a circular plasmid immobilized on paramagnetic beads is used as a template. DNA synthesis occurred on either circular or linear plasmids coupled to the beads, but only DNA synthesis on the circular plasmid was inhibited by geminin and a CDK inhibitor, p21. DNA synthesis on the circular plasmid occurred after a time lag, during which nuclear formation was probably occurring. Although pre-replicative complexes (pre-RCs) were formed soon after mixing plasmids with egg extracts, binding of CDC45, RPA, Pol α, δ and , and PCNA to the circular plasmid was delayed, but still correlated with DNA synthesis. Moreover, p21 inhibited binding of these replication fork proteins to the circular plasmid. Therefore, the circular plasmid, but not the linear plasmid, assembles bona fide replication forks in egg extracts. We conclude that this improved replication system will be useful for studying the mechanism of formation of replication forks in eukaryotic DNA replication.     

Fanconi anemia proteins stabilize replication forks

Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that chromatin binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.     

Stability of blocked replication forks in vivo

Replication of chromosomal DNA must be carried out to completion in order for a cell to proliferate. However, replication forks can stall during this process for a variety of reasons, including nucleoprotein ‘roadblocks’ and DNA lesions. In these circumstances the replisome copying the DNA may disengage from the chromosome to allow various repair processes to restore DNA integrity and enable replication to continue. Here, we report the in vivo stability of the replication fork when it encounters a nucleoprotein blockage in Escherichia coli. Using a site-specific and reversible protein block system in conjunction with the temperature sensitive DnaC helicase loader and DnaB replicative helicase, we monitored the disappearance of the Y-shaped DNA replication fork structures using neutral-neutral 2D agarose gels. We show the replication fork collapses within 5 min of encountering the roadblock. Therefore, the stalled replication fork does not pause at a block in a stable confirmation for an extended period of time as previously postulated.     

Interplay of replication checkpoints and repair proteins at stalled replication forks

DNA replication is an essential process that occurs in all growing cells and needs to be tightly regulated in order to preserve genetic integrity. Eukaryotic cells have developed multiple mechanisms to ensure the fidelity of replication and to coordinate the progression of replication forks. Replication is often impeded by DNA damage or replication blocks, and the resulting stalled replication forks are sensed and protected by specialized surveillance mechanisms called checkpoints. The replication checkpoint plays an essential role in preventing the breakdown of stalled replication forks and the accumulation of DNA structures that enhance recombination and chromosomal rearrangements that ultimately lead to genomic instability and cancer development. In addition, the replication checkpoint is thought to assist and coordinate replication fork restart processes by controlling DNA repair pathways, regulating chromatin structure, promoting the recruitment of proteins to sites of damage, and controlling cell cycle progression. In this review we focus mainly on the results obtained in budding yeast to discuss on the multiple roles of checkpoints in maintaining fork integrity and on the enzymatic activities that cooperate with the checkpoint pathway to promote fork resumption and repair of DNA lesions thereby contributing to genome integrity.     

Changes in the architecture and abundance of replication intermediates delineate the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remains elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.     

Recombination proteins and rescue of arrested replication forks

Recombination proteins play crucial roles in the rescue of inactivated replication forks in Escherichia coli. The enzymes that catalyze the repair of DNA double-strand breaks by a classical strand-exchange reaction (RecBCD, RecA) act in two well-characterized fork repair pathways. They repair the DNA double-strand end made when a replication fork runs into a single-strand interruption. They reset the DNA double-strand end generated by replication fork reversal when a component of the replication machinery is inactivated. In addition, recombination proteins also act at replication forks in ways other than the classical strand-exchange reaction. For example, the RuvAB enzyme that catalyzes Holliday junction branch-migration during homologous recombination is also able to catalyze replication fork reversal in certain replication mutants, i.e. to convert certain blocked replication forks into Holliday junctions. Finally, some of the actions of recombination proteins after replication impairment are still unclear, as for example in UV-irradiated cells, where RecFOR and RecA catalyze gap repair but also participate, in a yet undefined way, in "replisome reactivation".     

RuvAB is essential for replication forks reversal in certain replication mutants

Inactivated replication forks may be reversed by the annealing of leading- and lagging-strand ends, resulting in the formation of a Holliday junction (HJ) adjacent to a DNA double-strand end. In Escherichia coli mutants deficient for double-strand end processing, resolution of the HJ by RuvABC leads to fork breakage, a reaction that we can directly quantify. Here we used the HJ-specific resolvase RusA to test a putative role of the RuvAB helicase in replication fork reversal (RFR). We show that the RuvAB complex is required for the formation of a RusA substrate in the polymerase III mutants dnaEts and holD, affected for the Pol III catalytic subunit and clamp loader, and in the helicase mutant rep. This finding reveals that the recombination enzyme RuvAB targets forks in vivo and we propose that it directly converts forks into HJs. In contrast, RFR occurs in the absence of RuvAB in the dnaNts mutant, affected for the processivity clamp of Pol III, and in the priA mutant, defective for replication restart. This suggests alternative pathways of RFR.     

Recombinational repair and restart of damaged replication forks

Genome duplication necessarily involves the replication of imperfect DNA templates and, if left to their own devices, replication complexes regularly run into problems. The details of how cells overcome these replicative 'hiccups' are beginning to emerge, revealing a complex interplay between DNA replication, recombination and repair that ensures faithful passage of the genetic material from one generation to the next.     

Isolation of replication forks from growing Ehrlich ascites cells

A procedure is described which permits the large-scale isolation of essentially complete replications forks from the DNA of Ehrlich ascites cells. The whole nuclear DNA is first isolated by a method which involves minimal hydrodynamic shear. The DNA is then degraded by cryolysis, a freeze-thawing procedure, to a size providing the otherwise very labile forked structures with a sufficient resistance against shear forces. Finally, the Y-shaped structures of replicating DNA are separated by nitrocellulose column chromatography. When the newly formed strands of replicating DNA were density-labeled with 5-bromodeoxyuridine the DNA fraction isolated by this procedure banded in isopycnic CsCl gradients at a density expected for Y-shaped molecules with two light-heavy branches and one light-light branch and sedimented significantly faster than the corresponding bulk DNA fraction through neutral sucrose gradients. The forked molecules could be visualized by electron microscopy. The essential step of the procedure is the cryolysis which produces fragments from larger DNA structure essentially at random. When the cryolysis is omitted the forked structures are disrupted within the highly susceptible regions around the branching point.     

Collapse and repair of replication forks in Escherichia coli

Single-strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper-rec phenotype of the strains with increased numbers of single-strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat-mediated recombinational events and discuss a mechanism for quasi-conservative DNA replication explaining the recombinational repair-associated mutagenesis.     

Electron microscope study of a plasmid chimera containing the replication region of the Escherichia coli F plasmid.

pML31, a plasmid chimera constructed to contain the replication genes of an Flac plasmid, has been studied by electron microscope methods. Heteroduplex analysis shows that the only F sequence present in pML31 is that with corrdinates 40.3-49.3F. This region has previously been identified as essential for plasmid maintenance. The sequence of pML31, which was derived originally from R6-5, carries the km gene(s) and an inverted duplication of a 1.0-kilobase sequence. On the basis of length measurements, the repeated sequence is different from IS1, IS2, IS3, and an inverted repeat associated with the km gene(s) of plasmid JR67.     

DNA replication: Pif1 pulls the plug on stalled replication forks

The conserved PIF helicase family appears to function in replication to ensure termination and passage through regions that slow or arrest replication fork movement. Findings in fission yeast extend evidence from budding yeast, and argue for universal mechanisms that ensure replication integrity.     

Instability of inhibited replication forks in E. coli

Inhibiting the progress of replication forks in E. coli makes them susceptible to breakage. Broken replication forks are evidently reassembled by the RecBCD recombinational repair pathway. These findings explain a particular pattern of DNA degradation during inhibition of chromosomal replication, the role of recombination in the viability of mutants with displaced replication origin, and hyper-recombination observed in the Terminus of the E. coli chromosome in rnh mutants. Breakage and repair of inhibited replication forks could be the reason for the recombination-dependence of inducible stable DNA replication. A mechanism by which RecABCD-dependent recombination between very short inverted repeats may help E. coli to invert an operon, transcribed in the direction opposite to that of DNA replication, is discussed.     

The nonmutagenic repair of broken replication forks via recombination

When replication forks stall or collapse at sites of DNA damage, there are two avenues for fork rescue. Mutagenic translesion synthesis by a special class of DNA polymerases can move a fork past the damage, but can leave behind mutations. The alternative nonmutagenic pathways for fork repair involve cellular recombination systems. In bacteria, nonmutagenic repair of replication forks may occur as often as once per cell per generation, and is the favored path for fork restoration under normal growth conditions. Replication fork repair is almost certainly the major function of bacterial recombination systems, and was probably the impetus for the evolution of recombination systems. Increasingly, the nonmutagenic repair of replication forks is seen as a major function of eukaryotic recombination systems as well.