Replisome Stability Research Articles

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1–4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Mechanism of replication origin melting nucleated by CMG helicase assembly

The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45–MCM–GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown1. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.

DDK: The Outsourced Kinase of Chromosome Maintenance.

Simple SummaryTo ensure the maintenance of genetic stability prior to cell division a cell’s complement of chromosomes must be duplicated. This requires not only the replication of the chromosomal DNA but also the re-establishment the chromatin environment following duplication. To ensure the equal segregation of the genetic material to progeny cells, the duplicated chromatid pairs must remain physically coupled until cell division. The regulation of chromosome duplication is under the overall control of the cyclin-dependent kinases (CDK). In addition to maintaining global control of chromosome duplication, CDK directs the activation of a second kinase, the Dbf4-dependent kinase (DDK), which functions locally to facilitate the activation DNA replication and to coordinate this with the re-establishment of chromatin and the physical coupling of the chromatids following duplication. In this review, we discuss this ‘outsourcing’ by CDK to DDK of the activities that must be coordinated to ensure chromosome maintenance during cell division.The maintenance of genomic stability during the mitotic cell-cycle not only demands that the DNA is duplicated and repaired with high fidelity, but that following DNA replication the chromatin composition is perpetuated and that the duplicated chromatids remain tethered until their anaphase segregation. The coordination of these processes during S phase is achieved by both cyclin-dependent kinase, CDK, and Dbf4-dependent kinase, DDK. CDK orchestrates the activation of DDK at the G1-to-S transition, acting as the ‘global’ regulator of S phase and cell-cycle progression, whilst ‘local’ control of the initiation of DNA replication and repair and their coordination with the re-formation of local chromatin environments and the establishment of chromatid cohesion are delegated to DDK. Here, we discuss the regulation and the multiple roles of DDK in ensuring chromosome maintenance. Regulation of replication initiation by DDK has long been known to involve phosphorylation of MCM2-7 subunits, but more recent results have indicated that Treslin:MTBP might also be important substrates. Molecular mechanisms by which DDK regulates replisome stability and replicated chromatid cohesion are less well understood, though important new insights have been reported recently. We discuss how the ‘outsourcing’ of activities required for chromosome maintenance to DDK allows CDK to maintain outright control of S phase progression and the cell-cycle phase transitions whilst permitting ongoing chromatin replication and cohesion establishment to be completed and achieved faithfully.

Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress.

The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.

Acute hydroxyurea-induced replication blockade results in replisome components disengagement from nascent DNA without causing fork collapse.

During S phase, replication forks can encounter several obstacles that lead to fork stalling, which if persistent might result in fork collapse. To avoid this collapse and to preserve the competence to restart, cells have developed mechanisms that maintain fork stability upon replication stress. In this study, we aimed to understand the mechanisms involved in fork stability maintenance in non-transformed human cells by performing an isolation of proteins on nascent DNA-mass spectrometry analysis in hTERT-RPE cells under different replication stress conditions. Our results show that acute hydroxyurea-induced replication blockade causes the accumulation of large amounts of single-stranded DNA at the fork. Remarkably, this results in the disengagement of replisome components from nascent DNA without compromising fork restart. Notably, Cdc45-MCM-GINS helicase maintains its integrity and replisome components remain associated with chromatin upon acute hydroxyurea treatment, whereas replisome stability is lost upon a sustained replication stress that compromises the competence to restart.

MTOR Signaling Upregulates CDC6 via Suppressing miR-3178 and Promotes the Loading of DNA Replication Helicase

mTOR signaling pathway is deregulated in most cancers and uncontrolled cell cycle progression is a hallmark of cancer cell. However, the precise molecular mechanisms of the regulation of DNA replication and chromatin metabolism by mTOR signaling are largely unknown. We herein report that mTOR signaling promotes the loading of MCM2-7 helicase onto chromatin and upregulates DNA replication licensing factor CDC6. Pharmacological inhibition of mTOR kinase resulted in CHK1 checkpoint activation and decreased MCM2-7 replication helicase and PCNA associated with chromatins. Further pharmacological and genetic studies demonstrated CDC6 is positively controlled by mTORC1-S6K1 and mTORC2 signaling. miRNA screening revealed mTOR signaling suppresses miR-3178 thereby upregulating CDC6. Analysis of TCGA data found that CDC6 is overexpressed in most cancers and associates with the poor survival of cancer patients. Our findings suggest that mTOR signaling may control DNA replication origin licensing and replisome stability thereby cell cycle progression through CDC6 regulation.

TRAIP is a master regulator of DNA interstrand crosslink repair.

Summary:Cells often utilize multiple pathways to repair the same DNA lesion, and pathway choice has profound implications for the fidelity of genome maintenance. DNA interstrand cross-links (ICLs) block DNA replication and transcription by covalently linking the two strands of DNA, and the cytotoxicity of ICLs is exploited for chemotherapy. In Xenopus egg extracts, replication fork collision with ICLs initiates two distinct repair pathways. The NEIL3 glycosylase can cleave the cross-link1, but if this fails, the Fanconi anemia (FA) proteins incise the phosphodiester backbone surrounding the ICL, generating a double-strand break (DSB) intermediate that is repaired by homologous recombination2. How the simpler NEIL3 pathway is prioritized over the FA pathway, which can cause genomic rearrangements, is unknown. Here, we show that the E3 ubiquitin ligase TRAIP regulates both pathways. TRAIP appears to associate with the leading edge of the replisome, ubiquitylating any protein in the replisome’s path, including the replicative DNA helicase CMG (CDC45-MCM2-7-GINS) when two replisomes converge at an ICL. In this setting, short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for CMG unloading by the p97 ATPase, enabling the FA pathway. Our results identify TRAIP as a master regulator of replisome stability and ICL repair pathway choice.

Xrs2 and Tel1 Independently Contribute to MR-Mediated DNA Tethering and Replisome Stability

SUMMARYThe yeast Mre11-Rad50-Xrs2 (MRX) complex has structural, signaling, and catalytic functions in the response to DNA damage. Xrs2, the eukaryotic-specific component of the complex, is required for nuclear import of Mre11 and Rad50 and to recruit the Tel1 kinase to damage sites. We show that nuclear-localized MR complex (Mre11-NLS) catalyzes homology-dependent repair without Xrs2, but MR cannot activate Tel1, and it fails to tether DSBs, resulting in sensitivity to genotoxins, replisome instability, and increased gross chromosome rearrangements (GCRs). Fusing the Tel1 interaction domain from Xrs2 to Mre11-NLS is sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells. Furthermore, Tel1 stabilizes Mre11-DNA association, and this stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Enforcing Tel1 recruitment to the nuclear MR complex fully rescues end tethering and stalled replication fork stability, and suppresses GCRs, highlighting important roles for Xrs2 and Tel1 to ensure optimal MR activity.

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria.

Within the last decade, it has become clear that DNA replication and transcription are routinely in conflict with each other in growing cells. Much of the seminal work on this topic has been carried out in bacteria, specifically, Escherichia coli and Bacillus subtilis; therefore, studies of conflicts in these species deserve special attention. Collectively, the recent findings on conflicts have fundamentally changed the way we think about DNA replication in vivo. Furthermore, new insights on this topic have revealed that the conflicts between replication and transcription significantly influence many key parameters of cellular function, including genome organization, mutagenesis, and evolution of stress response and virulence genes. In this review, we discuss the consequences of replication-transcription conflicts on the life of bacteria and describe some key strategies cells use to resolve them. We put special emphasis on two critical aspects of these encounters: ( a) the consequences of conflicts on replisome stability and dynamics, and ( b) the resulting increase in spontaneous mutagenesis.

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

SummaryDbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation.

The Replication Checkpoint Prevents Two Types of Fork Collapse without Regulating Replisome Stability

The ATR replication checkpoint ensures that stalled forks remain stable when replisome movement is impeded. Using an improved iPOND protocol combined with SILAC mass spectrometry, we characterized human replisome dynamics in response to fork stalling. Our data provide a quantitative picture of the replisome and replication stress response proteomes in 32 experimental conditions. Importantly, rather than stabilize the replisome, the checkpoint prevents two distinct types of fork collapse. Unsupervised hierarchical clustering of protein abundance on nascent DNA is sufficient to identify protein complexes and place newly identified replisome-associated proteins into functional pathways. As an example, we demonstrate that ZNF644 complexes with the G9a/GLP methyltransferase at replication forks and is needed to prevent replication-associated DNA damage. Our data reveal how the replication checkpoint preserves genome integrity, provide insights into the mechanism of action of ATR inhibitors, and will be a useful resource for replication, DNA repair, and chromatin investigators.

A Rad53 Independent Function of Rad9 Becomes Crucial for Genome Maintenance in the Absence of the RecQ Helicase Sgs1

The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS) induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU)-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3). Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.

A Role for H2B Ubiquitylation in DNA Replication

The monoubiquitylation of histone H2B plays an important role in gene expression by contributing to the regulation of transcription elongation and mRNA processing and export. We explored additional cellular functions of this histone modification by investigating its localization to intergenic regions. H2B ubiquitylation is present in chromatin around origins of DNA replication in budding yeast, and as DNA is replicated its levels are maintained on daughter strands by the Bre1 ubiquitin ligase. In the absence of H2B ubiquitylation, the prereplication complex is formed and activated, but replication fork progression is slowed down and the replisome becomes unstable in the presence of hydroxyurea. H2B ubiquitylation promotes the assembly or stability of nucleosomes on newly replicated DNA, and this function is postulated to contribute to fork progression and replisome stability.

Replisome Stability at Defective DNA Replication Forks Is Independent of S Phase Checkpoint Kinases

The S phase checkpoint pathway preserves genome stability by protecting defective DNA replication forks, but the underlying mechanisms are still understood poorly. Previous work with budding yeast suggested that the checkpoint kinases Mec1 and Rad53 might prevent collapse of the replisome when nucleotide concentrations are limiting, thereby allowing the subsequent resumption of DNA synthesis. Here we describe a direct analysis of replisome stability in budding yeast cells lacking checkpoint kinases, together with a high-resolution view of replisome progression across the genome. Surprisingly, we find that the replisome is stably associated with DNA replication forks following replication stress in the absence of Mec1 or Rad53. A component of the replicative DNA helicase is phosphorylated within the replisome in a Mec1-dependent manner upon replication stress, and our data indicate that checkpoint kinases control replisome function rather than stability, as part of a multifaceted response that allows cells to survive defects in chromosome replication.

Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes

The eukaryotic replicative DNA polymerases (Pol α, δ, and ε), and the major DNA mutagenesis enzyme Pol ζ contain two conserved cysteine-rich metal-binding motifs (CysA and CysB) in the C-terminal domain (CTD) of their catalytic subunits. Here, we demonstrate by in vivo and in vitro approaches the presence of an essential [4Fe-4S] cluster in the CysB motif of all four yeast B-family DNA polymerases. Loss of the [4Fe-4S] cofactor by cysteine ligand mutagenesis in Pol3 destabilized the CTD and abrogated interaction with the Pol31-Pol32 subunits. Reciprocally, overexpression of accessory subunits increased the amount of CTD-bound Fe-S cluster. This implies an important physiological role of the Fe-S cluster in polymerase complex stabilization. Further, we demonstrate that the Zn-binding CysA motif is required for PCNA-mediated Pol δ processivity. Together, our findings show that the function of eukaryotic replicative DNA polymerases crucially depends on different metallocenters for accessory subunit recruitment and for replisome stability.

Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.