Eukaryotic Replisome Research Articles

How to write an ending: Telomere replication as a multistep process

Telomeres are protective nucleoprotein caps found at the natural ends of eukaryotic chromosomes and are crucial for the preservation of stable chromosomal structure. In cycling cells, telomeres are maintained by a multi-step process called telomere replication, which involves the eukaryotic replisome navigating a complex repetitive template tightly bound by specific proteins, before terminating at the chromosome end prior to a 5’ resection step that generates a protective 3’ overhang. In this review, we examine mechanistic aspects of the telomere replication process and consider how individual parts of this multistep event are integrated and coordinated with one-another.

USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination.

The CMG helicase (CDC45-MCM2-7-GINS) unwinds DNA as a component of eukaryotic replisomes. Replisome (dis)assembly is tightly coordinated with cell cycle progression to ensure genome stability. However, factors that prevent premature CMG unloading and replisome disassembly are poorly described. Since disassembly is catalyzed by ubiquitination, deubiquitinases (DUBs) represent attractive candidates for safeguarding against untimely and deleterious CMG unloading. We combined a targeted loss-of-function screen with quantitative, single-cell analysis to identify human USP37 as a key DUB preventing replisome disassembly. We demonstrate that USP37 maintains active replisomes on S-phase chromatin and promotes normal cell cycle progression. Proteomics and enzyme assays revealed USP37 interacts with the CMG complex to deubiquitinate MCM7, thus antagonizing replisome disassembly. Significantly, USP37 protects normal epithelial cells from oncoprotein-induced replication stress. Our findings reveal USP37 to be critical to the maintenance of replisomes in S-phase and suggest USP37-targeting as a potential strategy for treating malignancies with defective DNA replication control.

A common mechanism for recruiting the Rrm3 and RTEL1 accessory helicases to the eukaryotic replisome

The eukaryotic replisome is assembled around the CMG (CDC45-MCM-GINS) replicative helicase, which encircles the leading-strand DNA template at replication forks. When CMG stalls during DNA replication termination, or at barriers such as DNA-protein crosslinks on the leading strand template, a second helicase is deployed on the lagging strand template to support replisome progression. How these ‘accessory’ helicases are targeted to the replisome to mediate barrier bypass and replication termination remains unknown. Here, by combining AlphaFold structural modelling with experimental validation, we show that the budding yeast Rrm3 accessory helicase contains two Short Linear Interaction Motifs (SLIMs) in its disordered N-terminus, which interact with CMG and the leading-strand DNA polymerase Polε on one side of the replisome. This flexible tether positions Rrm3 adjacent to the lagging strand template on which it translocates, and is critical for replication termination in vitro and Rrm3 function in vivo. The primary accessory helicase in metazoa, RTEL1, is evolutionarily unrelated to Rrm3, but binds to CMG and Polε in an analogous manner, revealing a conserved docking mechanism for accessory helicases in the eukaryotic replisome.

CMG helicase disassembly is essential and driven by two pathways in budding yeast

The CMG helicase is the stable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, suggesting that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCFDia2. mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2∆ is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes.

The Origin Recognition Complex: From Origin Selection to Replication Licensing in Yeast and Humans.

Understanding human DNA replication through the study of yeast has been an extremely fruitful journey. The minichromosome maintenance (MCM) 2-7 genes that encode the catalytic core of the eukaryotic replisome were initially identified through forward yeast genetics. The origin recognition complexes (ORC) that load the MCM hexamers at replication origins were purified from yeast extracts. We have reached an age where high-resolution cryoEM structures of yeast and human replication complexes can be compared side-by-side. Their similarities and differences are converging as alternative strategies that may deviate in detail but are shared by both species.

DONSON is required for CMG helicase assembly in the mammalian cell cycle.

DONSON is one of 13 genes mutated in a form of primordial microcephalic dwarfism known as Meier-Gorlin syndrome. The other 12 encode components of the CDC45-MCM-GINS helicase, around which the eukaryotic replisome forms, or are factors required for helicase assembly during DNA replication initiation. A role for DONSON in CDC45-MCM-GINS assembly was unanticipated, since DNA replication initiation can be reconstituted in vitro with purified proteins from budding yeast, which lacks DONSON. Using mouse embryonic stem cells as a model for the mammalian helicase, we show that DONSON binds directly but transiently to CDC45-MCM-GINS during S-phase and is essential for chromosome duplication. Rapid depletion of DONSON leads to the disappearance of the CDC45-MCM-GINS helicase from S-phase cells and our data indicate that DONSON is dispensable for loading of the MCM2-7 helicase core onto chromatin during G1-phase, but instead is essential for CDC45-MCM-GINS assembly during S-phase. These data identify DONSON as a missing link in our understanding of mammalian chromosome duplication and provide a molecular explanation for why mutations in human DONSON are associated with Meier-Gorlin syndrome.

DNA replication machineries: Structural insights from crystallography and electron microscopy.

Since the discovery of DNA as the genetic material, scientists have been investigating how the information contained in this biological polymer is transmitted from generation to generation. X-ray crystallography, and more recently, cryo-electron microscopy techniques have been instrumental in providing essential information about the structure, functions and interactions of the DNA and the protein machinery (replisome) responsible for its replication. In this chapter, we highlight several works that describe the structure and structure-function relationships of the core components of the prokaryotic and eukaryotic replisomes. We also discuss the most recent studies on the structural organization of full replisomes.

The mechanism of replication stalling and recovery within repetitive DNA

Accurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstitute replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We find that structure-prone repeats are sufficient to impair replication. Whilst template unwinding is unaffected, leading strand synthesis is inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats is rescued by replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats requires an extrinsic accessory helicase. DNA-induced fork stalling is mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress may also be applied to DNA-induced replication stalling.

The Fork Protection Complex: A Regulatory Hub at the Head of the Replisome.

As well as accurately duplicating DNA, the eukaryotic replisome performs a variety of other crucial tasks to maintain genomic stability. For example, organizational elements, like cohesin, must be transferred from the front of the fork to the new strands, and when there is replication stress, forks need to be protected and checkpoint signalling activated. The Tof1-Csm3 (or Timeless-Tipin in humans) Fork Protection Complex (FPC) ensures efficient replisome progression and is required for a range of replication-associated activities. Recent studies have begun to reveal the structure of this complex, and how it functions within the replisome to perform its diverse roles. The core of the FPC acts as a DNA grip on the front of the replisome to regulate fork progression. Other flexibly linked domains and motifs mediate interactions with proteins and specific DNA structures, enabling the FPC to act as a hub at the head of the replication fork.

TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.

Mechanisms for Maintaining Eukaryotic Replisome Progression in the Presence of DNA Damage.

The eukaryotic replisome coordinates template unwinding and nascent-strand synthesis to drive DNA replication fork progression and complete efficient genome duplication. During its advancement along the parental template, each replisome may encounter an array of obstacles including damaged and structured DNA that impede its progression and threaten genome stability. A number of mechanisms exist to permit replisomes to overcome such obstacles, maintain their progression, and prevent fork collapse. A combination of recent advances in structural, biochemical, and single-molecule approaches have illuminated the architecture of the replisome during unperturbed replication, rationalised the impact of impediments to fork progression, and enhanced our understanding of DNA damage tolerance mechanisms and their regulation. This review focusses on these studies to provide an updated overview of the mechanisms that support replisomes to maintain their progression on an imperfect template.

Unchecked nick ligation can promote localized genome re-replication

SummarySingle-stranded DNA breaks, or nicks, are amongst the most common forms of DNA damage in cells. They can be repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a collapsed replication fork comprising a single-ended double-strand break and a ‘hybrid nick’ with parental DNA on one side and nascent DNA on the other (Figure 1A). We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a fork from an adjacent origin can promote localized re-replication if the hybrid nick is ligated. We have modelled this situation with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated.

Budding yeast Rap1, but not telomeric DNA, is inhibitory for multiple stages of DNA replication in vitro.

Telomeres are copied and reassembled each cell division cycle through a multistep process called telomere replication. Most telomeric DNA is duplicated semiconservatively during this process, but replication forks frequently pause or stall at telomeres in yeast, mouse and human cells, potentially causing chronic telomere shortening or loss in a single cell cycle. We have investigated the cause of this effect by examining the replication of telomeric templates in vitro. Using a reconstituted assay for eukaryotic DNA replication in which a complete eukaryotic replisome is assembled and activated with purified proteins, we show that budding yeast telomeric DNA is efficiently duplicated in vitro unless the telomere binding protein Rap1 is present. Rap1 acts as a roadblock that prevents replisome progression and leading strand synthesis, but also potently inhibits lagging strand telomere replication behind the fork. Both defects can be mitigated by the Pif1 helicase. Our results suggest that GC-rich sequences do not inhibit DNA replication per se, and that in the absence of accessory factors, telomere binding proteins can inhibit multiple, distinct steps in the replication process.

Increased expression of Polδ does not alter the canonical replication program in vivo.

Background: In vitro experiments utilising the reconstituted Saccharomyces cerevisiae eukaryotic replisome indicated that the efficiency of the leading strand replication is impaired by a moderate increase in Polδ concentration. It was hypothesised that the slower rate of the leading strand synthesis characteristic for reactions containing two-fold and four-fold increased concentration of Polδ represented a consequence of a relatively rare event, during which Polδ stochastically outcompeted Polε and, in an inefficient manner, temporarily facilitated extension of the leading strand. Inspired by this observation, we aimed to determine whether similarly increased Polδ levels influence replication dynamics in vivo using the fission yeast Schizosaccharomyces pombe as a model system. Methods: To generate S. pombe strains over-expressing Polδ, we utilised Cre-Lox mediated cassette exchange and integrated one or three extra genomic copies of all four Polδ genes. To estimate expression of respective Polδ genes in Polδ-overexpressing mutants, we measured relative transcript levels of cdc1 + , cdc6 + (or cdc6 L591G ), cdc27 + and cdm1 + by reverse transcription followed by quantitative PCR (RT-qPCR). To assess the impact of Polδ over-expression on cell physiology and replication dynamics, we used standard cell biology techniques and polymerase usage sequencing. Results: We provide an evidence that two-fold and four-fold over-production of Polδ does not significantly alter growth rate, cellular morphology and S-phase duration. Polymerase usage sequencing analysis further indicates that increased Polδ expression does not change activities of Polδ, Polε and Polα at replication initiation sites and across replication termination zones. Additionally, we show that mutants over-expressing Polδ preserve WT-like distribution of replication origin efficiencies. Conclusions: Our experiments do not disprove the existence of opportunistic polymerase switches; however, the data indicate that, if stochastic replacement of Polε for Polδ does occur i n vivo, it represents a rare phenomenon that does not significantly influence canonical replication program.

Increased expression of Polδ does not alter the canonical replication program in vivo

Background: In vitro experiments utilising the reconstituted Saccharomyces cerevisiae eukaryotic replisome indicated that the efficiency of the leading strand replication is impaired by a moderate increase in Polδ concentration. It was hypothesised that the slower rate of the leading strand synthesis characteristic for reactions containing two-fold and four-fold increased concentration of Polδ represented a consequence of a relatively rare event, during which Polδ stochastically outcompeted Polε and, in an inefficient manner, temporarily facilitated extension of the leading strand. Inspired by this observation, we aimed to determine whether similarly increased Polδ levels influence replication dynamics in vivo using the fission yeast Schizosaccharomyces pombe as a model system. Methods: To generate S. pombe strains over-expressing Polδ, we utilised Cre-Lox mediated cassette exchange and integrated one or three extra genomic copies of all four Polδ genes. To estimate expression of respective Polδ genes in Polδ-overexpressing mutants, we measured relative transcript levels of cdc1 +, cdc6 + (or cdc6 L591G), cdc27 + and cdm1 + by reverse transcription followed by quantitative PCR (RT-qPCR). To assess the impact of Polδ over-expression on cell physiology and replication dynamics, we used standard cell biology techniques and polymerase usage sequencing. Results: We provide an evidence that two-fold and four-fold over-production of Polδ does not significantly alter growth rate, cellular morphology and S-phase duration. Polymerase usage sequencing analysis further indicates that increased Polδ expression does not change activities of Polδ, Polε and Polα at replication initiation sites and across replication termination zones. Additionally, we show that mutants over-expressing Polδ preserve WT-like distribution of replication origin efficiencies. Conclusions: Our experiments do not disprove the existence of opportunistic polymerase switches; however, the data indicate that, if stochastic replacement of Polε for Polδ does occur i n vivo, it represents a rare phenomenon that does not significantly influence canonical replication program.

DNA polymerase D temporarily connects primase to the CMG-like helicase before interacting with proliferating cell nuclear antigen.

The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and Polϵ in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of how it alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD–primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.

Increased expression of Polδ does not alter the canonical replication program in vivo.

Background: In vitro experiments utilising the reconstituted Saccharomyces cerevisiae eukaryotic replisome indicated that the efficiency of the leading strand replication is impaired by a moderate increase in Polδ concentration. It was hypothesised that the slower rate of the leading strand synthesis characteristic for reactions containing two-fold and four-fold increased concentration of Polδ represented a consequence of a relatively rare event, during which Polδ stochastically outcompeted Polε and, in an inefficient manner, temporarily facilitated extension of the leading strand. Inspired by this observation, we aimed to determine whether similarly increased Polδ levels influence replication dynamics in vivo using the fission yeast Schizosaccharomyces pombe as a model system. Methods: To generate S. pombe strains over-expressing Polδ, we utilised Cre-Lox mediated cassette exchange and integrated one or three extra genomic copies of all four Polδ genes. To estimate expression of respective Polδ genes in Polδ-overexpressing mutants, we measured relative transcript levels of cdc1 + , cdc6 + (or cdc6 L591G ), cdc27 + and cdm1 + by reverse transcription followed by quantitative PCR (RT-qPCR). To assess the impact of Polδ over-expression on cell physiology and replication dynamics, we used standard cell biology techniques and polymerase usage sequencing. Results: We provide an evidence that two-fold and four-fold over-production of Polδ does not significantly alter growth rate, cellular morphology and S-phase duration. Polymerase usage sequencing analysis further indicates that increased Polδ expression does not change activities of Polδ, Polε and Polα at replication initiation sites and across replication termination zones. Additionally, we show that mutants over-expressing Polδ preserve WT-like distribution of replication origin efficiencies. Conclusions: Our experiments do not disprove the existence of opportunistic polymerase switches; however, the data indicate that, if stochastic replacement of Polε for Polδ does occur i n vivo, it represents a rare phenomenon that does not significantly influence canonical replication program.

CUL2LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle.

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG‐MCM7 subunit during S‐phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis‐regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.

The eukaryotic replisome tolerates leading‐strand base damage by replicase switching

The high‐fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low‐fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading‐strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase–polymerase uncoupling a switch from Pol ε, the canonical leading‐strand replicase, to the lagging‐strand replicase Pol δ, facilitates rapid, efficient and error‐free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8‐oxoguanine. We propose that replicase switching may promote continued leading‐strand synthesis whenever the replisome encounters leading‐strand damage that is bypassed more efficiently by Pol δ than by Pol ε.

Anatomy of a twin DNA replication factory.

The replication of DNA in chromosomes is initiated at sequences called origins at which two replisome machines are assembled at replication forks that move in opposite directions. Interestingly, in vivo studies observe that the two replication forks remain fastened together, often referred to as a replication factory. Replication factories containing two replisomes are well documented in cellular studies of bacteria (Escherichia coli and Bacillus subtilis) and the eukaryote, Saccharomyces cerevisiae. This basic twin replisome factory architecture may also be preserved in higher eukaryotes. Despite many years of documenting the existence of replication factories, the molecular details of how the two replisome machines are tethered together has been completely unknown in any organism. Recent structural studies shed new light on the architecture of a eukaryote replisome factory, which brings with it a new twist on how a replication factory may function.