Eukaryotic DNA replication. Enzymes and proteins acting at the fork

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. model systems for replication, such as the simian virus 40 DNA, lead the way. Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double-helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase delta and the lagging strand discontinuously in small pieces by DNA polymerase alpha. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3'----5'-exonuclease, mostly part of the DNA polymerase delta polypeptide, can perform proof-reading by excising incorrectly base-paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5'----3'-exonuclease, removing the RNA primers, DNA polymerase alpha or beta, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher-order structures like chromatin.     

Proteolytic control of genome integrity at the replication fork

Faithful duplication of the genome is critical for the survival of an organism and prevention of malignant transformation. Accurate replication of a large amount of genetic information in a timely manner is one of the most challenging cellular processes and is often perturbed by intrinsic and extrinsic barriers to DNA replication fork progression, a phenomenon referred to as DNA replication stress. Elevated DNA replication stress is a primary source of genomic instability and one of the key hallmarks of cancer. Therefore, targeting DNA replication stress is an emerging concept for cancer therapy. The replication machinery associated with PCNA and other regulatory factors coordinates the synthesis and repair of DNA strands at the replication fork. The dynamic interaction of replication protein complexes with DNA is essential for sensing and responding to various signaling events relevant to DNA replication and damage. Thus, the disruption of the spatiotemporal regulation of protein homeostasis at the replication fork impairs genome integrity, which often involves the deregulation of ubiquitin-mediated proteolytic signaling. Notably, emerging evidence has highlighted the role of the AAA+ATPase VCP/p97 in extracting ubiquitinated protein substrates from the chromatin and facilitating the turnover of genome surveillance factors during DNA replication and repair. Here, we review recent advances in our understanding of chromatin-associated degradation pathways at the replication fork and the implication of these findings for cancer therapy.     

DNA polymerases at the replication fork in eukaryotes

The Kunkel laboratory has recently assigned polymerase (Pol) epsilon as the leading strand polymerase. In a recent issue of Molecular Cell, they now assign Pol delta as the lagging strand polymerase.     

Polymerase dynamics at the eukaryotic DNA replication fork

This review discusses recent insights in the roles of DNA polymerases (Pol) delta and epsilon in eukaryotic DNA replication. A growing body of evidence specifies Pol epsilon as the leading strand DNA polymerase and Pol delta as the lagging strand polymerase during undisturbed DNA replication. New evidence supporting this model comes from the use of polymerase mutants that show an asymmetric mutator phenotype for certain mispairs, allowing an unambiguous strand assignment for these enzymes. On the lagging strand, Pol delta corrects errors made by Pol alpha during Okazaki fragment initiation. During Okazaki fragment maturation, the extent of strand displacement synthesis by Pol delta determines whether maturation proceeds by the short or long flap processing pathway. In the more common short flap pathway, Pol delta coordinates with the flap endonuclease FEN1 to degrade initiator RNA, whereas in the long flap pathway, RNA removal is initiated by the Dna2 nuclease/helicase.     

Mechanisms of replication fork protection: a safeguard for genome stability

During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.     

Mechanisms of replication fork protection: a safeguard for genome stability

During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.     

RNF4 controls the extent of replication fork reversal to preserve genome stability

Replication fork reversal occurs via a two-step process that entails reversal initiation and reversal extension. DNA topoisomerase IIalpha (TOP2A) facilitates extensive fork reversal, on one hand through resolving the topological stress generated by the initial reversal, on the other hand via its role in recruiting the SUMO-targeted DNA translocase PICH to stalled forks in a manner that is dependent on its SUMOylation by the SUMO E3 ligase ZATT. However, how TOP2A activities at stalled forks are precisely regulated remains poorly understood. Here we show that, upon replication stress, the SUMO-targeted ubiquitin E3 ligase RNF4 accumulates at stalled forks and targets SUMOylated TOP2A for ubiquitination and degradation. Downregulation of RNF4 resulted in aberrant activation of the ZATT–TOP2A–PICH complex at stalled forks, which in turn led to excessive reversal and elevated frequencies of fork collapse. These results uncover a previously unidentified regulatory mechanism that regulates TOP2A activities at stalled forks and thus the extent of fork reversal.     

DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier

DNA double-strand breaks (DSB) were shown to occur at the replication fork barrier in the ribosomal DNA of Saccharomyces cerevisiae using 2D-gel electrophoresis. Their origin, nature and magnitude, however, have remained elusive. We quantified these DSBs and show that a surprising 14% of replicating ribosomal DNA molecules are broken at the replication fork barrier in replicating wild-type cells. This translates into an estimated steady-state level of 7–10 DSBs per cell during S-phase. Importantly, breaks detectable in wild-type and sgs1 mutant cells differ from each other in terms of origin and repair. Breaks in wild-type, which were previously reported as DSBs, are likely an artefactual consequence of nicks nearby the rRFB. Sgs1 deficient cells, in which replication fork stability is compromised, reveal a class of DSBs that are detectable only in the presence of functional Dnl4. Under these conditions, Dnl4 also limits the formation of extrachromosomal ribosomal DNA circles. Consistently, dnl4 cells displayed altered fork structures at the replication fork barrier, leading us to propose an as yet unrecognized role for Dnl4 in the maintenance of ribosomal DNA stability.     

Metabolism of DNA secondary structures at the eukaryotic replication fork

DNA secondary structures are largely advantageous for numerous cellular processes but can pose specific threats to the progression of the replication machinery and therefore genome duplication and cell division. A number of specialized enzymes dismantle these structures to allow replication fork progression to proceed faithfully. In this review, we discuss the in vitro and in vivo data that has lead to the identification of these enzymes in eukaryotes, and the evidence that suggests that they act specifically at replication forks to resolve secondary structures. We focus on the role of helicases, which catalyze the dissociation of nucleotide complexes, and on the role of nucleases, which cleave secondary structures to allow replication fork progression at the expense of local rearrangements. Finally, we discuss outstanding questions in terms of dismantling DNA secondary structures, as well as the interplay between diverse enzymes that act upon specific types of structures.     

Oligomerization of DNA replication regulatory protein RADX is essential to maintain replication fork stability

Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. Many regulatory proteins control these RAD51 activities, including RADX, which binds both ssDNA and RAD51 at replication forks to ensure that fork reversal is confined to stalled forks. Many ssDNA-binding proteins function as hetero- or homo-oligomers. In this study, we addressed whether this is also the case for RADX. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.     

Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins

Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins"). These bobbins constitute a major portion of the mass of the full replication complex.     

Chromatin assembly controls replication fork stability

During DNA replication, the advance of replication forks is tightly connected with chromatin assembly, a process that can be impaired by the partial depletion of histone H4 leading to recombinogenic DNA damage. Here, we show that the partial depletion of H4 is rapidly followed by the collapse of unperturbed and stalled replication forks, even though the S‐phase checkpoints remain functional. This collapse is characterized by a reduction in the amount of replication intermediates, but an increase in single Ys relative to bubbles, defects in the integrity of the replisome and an accumulation of DNA double‐strand breaks. This collapse is also associated with an accumulation of Rad52‐dependent X‐shaped molecules. Consistently, a Rad52‐dependent—although Rad51‐independent—mechanism is able to rescue these broken replication forks. Our findings reveal that correct nucleosome deposition is required for replication fork stability, and provide molecular evidence for homologous recombination as an efficient mechanism of replication fork restart.     

Repair and tolerance of DNA damage at the replication fork: A structural perspective

The replication machinery frequently encounters DNA damage and other structural impediments that inhibit progression of the replication fork. Replication-coupled processes that remove or bypass the barrier and restart stalled forks are essential for completion of replication and for maintenance of genome stability. Errors in replication-repair pathways lead to mutations and aberrant genetic rearrangements and are associated with human diseases. This review highlights recent structures of enzymes involved in three replication-repair pathways: translesion synthesis, template switching and fork reversal, and interstrand crosslink repair.     

FANCD2 influences replication fork processes and genome stability in response to clustered DSBs

Fanconi Anemia (FA) is a cancer predisposition syndrome and the factors defective in FA are involved in DNA replication, DNA damage repair and tumor suppression. Here, we show that FANCD2 is critical for genome stability maintenance in response to high-linear energy transfer (LET) radiation. We found that FANCD2 is monoubiquitinated and recruited to the sites of clustered DNA double-stranded breaks (DSBs) specifically in S/G2 cells after high-LET radiation. Further, FANCD2 facilitated the repair of clustered DSBs in S/G2 cells and proper progression of S-phase. Furthermore, lack of FANCD2 led to a reduced rate of replication fork progression and elevated levels of both replication fork stalling and new origin firing in response to high-LET radiation. Mechanistically, FANCD2 is required for correct recruitment of RPA2 and Rad51 to the sites of clustered DSBs and that is critical for proper processing of clustered DSBs. Significantly, FANCD2-decifient cells exhibited defective chromosome segregation, elevated levels of chromosomal aberrations, and anchorage-independent growth in response to high-LET radiation. These findings establish FANCD2 as a key factor in genome stability maintenance in response to high-LET radiation and as a promising target to improve cancer therapy.     

Non-replicative helicases at the replication fork

Reactivation of stalled or collapsed replication forks is an essential process in bacteria. Restart systems operate to restore the 5'-->3' replicative helicase, DnaB, to the lagging-strand template. However, other non-replicative 3'-->5' helicases play an important role in the restart process as well. Here we examine the DNA-binding specificity of three of the latter group, PriA, Rep, and UvrD. Only PriA and Rep display structure-specific fork binding. Interestingly, their specificity is opposite: PriA binds a leading-strand fork, presumably reflecting its restart activity in directing loading of DnaB to the lagging-strand template. Rep binds a lagging-strand fork, presumably reflecting its role in partially displacing Okazaki fragments that originate near the fork junction. This activity is necessary for generating a single-stranded landing pad for DnaB. While UvrD shows little structure-specificity, there is a slight preference for lagging-strand forks, suggesting that there might be some redundancy between Rep and UvrD and possibly explaining the observed synthetic lethality that occurs when mutations in the genes encoding these two proteins are combined.     

The emerging determinants of replication fork stability

A universal response to replication stress is replication fork reversal, where the nascent complementary DNA strands are annealed to form a protective four-way junction allowing forks to avert DNA damage while replication stress is resolved. However, reversed forks are in turn susceptible to nucleolytic digestion of the regressed nascent DNA arms and rely on dedicated mechanisms to protect their integrity. The most well studied fork protection mechanism involves the BRCA pathway and its ability to catalyze RAD51 nucleofilament formation on the reversed arms of stalled replication forks. Importantly, the inability to prevent the degradation of reversed forks has emerged as a hallmark of BRCA deficiency and underlies genome instability and chemosensitivity in BRCA-deficient cells. In the past decade, multiple factors underlying fork stability have been discovered. These factors either cooperate with the BRCA pathway, operate independently from it to augment fork stability in its absence, or act as enablers of fork degradation. In this review, we examine these novel determinants of fork stability, explore the emergent conceptual underpinnings underlying fork protection, as well as the impact of fork protection on cellular viability and cancer therapy.     

DNA polymerases that propagate the eukaryotic DNA replication fork

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase ot-primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase ca and recruits DNA polymerase S and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase 8, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase E normally replicates this strand,but under conditions of dysfunction, DNA polymerase 8 may substitute.     

RMI1 promotes DNA replication fork progression and recovery from replication fork stress

RMI1 is a member of an evolutionarily conserved complex composed of BLM and topoisomerase IIIα (TopoIIIα). This complex exhibits strand passage activity in vitro, which is likely important for DNA repair and DNA replication in vivo. The inactivation of RMI1 causes genome instability, including elevated levels of sister chromatid exchange and accelerated tumorigenesis. Using molecular combing to analyze DNA replication at the single-molecule level, we show that RMI1 is required to promote normal replication fork progression. The fork progression defect in RMI1-depleted cells is alleviated in cells lacking BLM, indicating that RMI1 functions downstream of BLM in promoting replication elongation. RMI1 localizes to subnuclear foci with BLM and TopoIIIα in response to replication stress. The proper localization of the complex requires a BLM-TopoIIIα-RMI1 interaction and is essential for RMI1 to promote recovery from replication stress. These findings reveal direct roles of RMI1 in DNA replication and the replication stress response, which could explain the molecular basis for its involvement in suppressing sister chromatid exchange and tumorigenesis.