Leading Strand DNA Polymerase Research Articles

Structural basis for processive daughter-strand synthesis and proofreading by the human leading-strand DNA polymerase Pol ε.

During chromosome replication, the nascent leading strand is synthesized by DNA polymerase epsilon (Pol ε), which associates with the sliding clamp processivity factor proliferating cell nuclear antigen (PCNA) to form a processive holoenzyme. For high-fidelity DNA synthesis, Pol ε relies on nucleotide selectivity and its proofreading ability to detect and excise a misincorporated nucleotide. Here, we present cryo-electron microscopy (cryo-EM) structures of human Pol ε in complex with PCNA, DNA and an incoming nucleotide, revealing how Pol ε associates with PCNA through its PCNA-interacting peptide box and additional unique features of its catalytic domain. Furthermore, by solving a series of cryo-EM structures of Pol ε at a mismatch-containing DNA, we elucidate how Pol ε senses and edits a misincorporated nucleotide. Our structures delineate steps along an intramolecular switching mechanism between polymerase and exonuclease activities, providing the basis for a proofreading mechanism in B-family replicative polymerases.

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.

Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique β-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.

Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands.

Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.

Synergism between CMG helicase and leading strand DNA polymerase at replication fork

The replisome that replicates the eukaryotic genome consists of at least three engines: the Cdc45-MCM-GINS (CMG) helicase that separates duplex DNA at the replication fork and two DNA polymerases, one on each strand, that replicate the unwound DNA. Here, we determined a series of cryo-electron microscopy structures of a yeast replisome comprising CMG, leading-strand polymerase Polε and three accessory factors on a forked DNA. In these structures, Polε engages or disengages with the motor domains of the CMG by occupying two alternative positions, which closely correlate with the rotational movement of the single-stranded DNA around the MCM pore. During this process, the polymerase remains stably coupled to the helicase using Psf1 as a hinge. This synergism is modulated by a concerted rearrangement of ATPase sites to drive DNA translocation. The Polε-MCM coupling is not only required for CMG formation to initiate DNA replication but also facilitates the leading-strand DNA synthesis mediated by Polε. Our study elucidates a mechanism intrinsic to the replisome that coordinates the activities of CMG and Polε to negotiate any roadblocks, DNA damage, and epigenetic marks encountered during translocation along replication forks.

Symmetric inheritance of parental histones contributes to safeguarding the fate of mouse embryonic stem cells during differentiation.

Parental histones, the carriers of posttranslational modifications, are deposited evenly onto the replicating DNA of sister chromatids in a process dependent on the Mcm2 subunit of DNA helicase and the Pole3 subunit of leading-strand DNA polymerase. The biological significance of parental histone propagation remains unclear. Here we show that Mcm2-mutated or Pole3-deleted mouse embryonic stem cells (ESCs) display aberrant histone landscapes and impaired neural differentiation. Mutation of the Mcm2 histone-binding domain causes defects in pre-implantation development and embryonic lethality. ESCs with biased parental histone transfer exhibit increased epigenetic heterogeneity, showing altered histone variant H3.3 and H3K27me3 patterning at genomic sites regulating differentiation genes. Our results indicate that the lagging strand pattern of H3.3 leads to the redistribution of H3K27me3 in Mcm2-2A ESCs. We demonstrate that symmetric parental histone deposition to sister chromatids contributes to cellular differentiation and development.

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands.

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

Balancing act of a leading strand DNA polymerase-specific domain and its exonuclease domain promotes genome-wide sister replication fork symmetry.

Pol2 is the leading-strand DNA polymerase in budding yeast. Here we describe an antagonism between its conserved POPS (Pol2 family-specific catalytic core peripheral subdomain) and exonuclease domain and the importance of this antagonism in genome replication. We show that multiple defects caused by POPS mutations, including impaired growth and DNA synthesis, genome instability, and reliance on other genome maintenance factors, were rescued by exonuclease inactivation. Single-molecule data revealed that the rescue stemmed from allowing sister replication forks to progress at equal rates. Our data suggest that balanced activity of Pol2's POPS and exonuclease domains is vital for genome replication and stability.

H3K4me3 recognition by the COMPASS complex facilitates the restoration of this histone mark following DNA replication.

During DNA replication, parental H3-H4 marked by H3K4me3 are transferred almost equally onto leading and lagging strands of DNA replication forks. Mutations in replicative helicase subunit, Mcm2 (Mcm2-3A), and leading strand DNA polymerase subunit, Dpb3 (dpb3∆), result in asymmetric distributions of H3K4me3 at replicating DNA strands immediately following DNA replication. Here, we show that mcm2-3A and dpb3∆ mutant cells markedly reduce the asymmetric distribution of H3K4me3 during cell cycle progression before mitosis. Furthermore, the restoration of a more symmetric distribution of H3K4me3 at replicating DNA strands in these mutant cells is driven by methylating nucleosomes without H3K4me3 by the H3K4 methyltransferase complex, COMPASS. Last, both gene transcription machinery and the binding of parental H3K4me3 by Spp1 subunit of the COMPASS complex help recruit the enzyme to chromatin for the restoration of the H3K4me3-marked state following DNA replication, shedding light on inheritance of this mark following DNA replication.

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28.

Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.

Recombination and Pol ζ Rescue Defective DNA Replication upon Impaired CMG Helicase-Pol ε Interaction.

The CMG complex (Cdc45, Mcm2–7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.

Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication.

G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

Molecular mechanisms of eukaryotic origin initiation, replication fork progression, and chromatin maintenance.

Eukaryotic DNA replication is a highly dynamic and tightly regulated process. Replication involves several dozens of replication proteins, including the initiators ORC and Cdc6, replicative CMG helicase, DNA polymerase α-primase, leading-strand DNA polymerase ε, and lagging-strand DNA polymerase δ. These proteins work together in a spatially and temporally controlled manner to synthesize new DNA from the parental DNA templates. During DNA replication, epigenetic information imprinted on DNA and histone proteins is also copied to the daughter DNA to maintain the chromatin status. DNA methyltransferase 1 is primarily responsible for copying the parental DNA methylation pattern into the nascent DNA. Epigenetic information encoded in histones is transferred via a more complex and less well-understood process termed replication-couple nucleosome assembly. Here, we summarize the most recent structural and biochemical insights into DNA replication initiation, replication fork elongation, chromatin assembly and maintenance, and related regulatory mechanisms.

Two components of DNA replication-dependent LexA cleavage

Induction of the SOS response, a cellular system triggered by DNA damage in bacteria, depends on DNA replication for the generation of the SOS signal, ssDNA. RecA binds to ssDNA, forming filaments that stimulate proteolytic cleavage of the LexA transcriptional repressor, allowing expression of > 40 gene products involved in DNA repair and cell cycle regulation. Here, using a DNA replication system reconstituted in vitro in tandem with a LexA cleavage assay, we studied LexA cleavage during DNA replication of both undamaged and base-damaged templates. Only a ssDNA-RecA filament supported LexA cleavage. Surprisingly, replication of an undamaged template supported levels of LexA cleavage like that induced by a template carrying two site-specific cyclobutane pyrimidine dimers. We found that two processes generate ssDNA that could support LexA cleavage. 1) During unperturbed replication, single-stranded regions formed because of stochastic uncoupling of the leading-strand DNA polymerase from the replication fork DNA helicase, and 2) on the damaged template, nascent leading-strand gaps were generated by replisome lesion skipping. The two pathways differed in that RecF stimulated LexA cleavage during replication of the damaged template, but not normal replication. RecF appears to facilitate RecA filament formation on the leading-strand ssDNA gaps generated by replisome lesion skipping.

Structure of the polymerase \u03b5 holoenzyme and atomic model of the leading strand replisome

The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ε holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ε-PCNA clamp. We show here a 3.5-Å cryo-EM structure of yeast Pol ε revealing that the Dpb3–Dpb4 subunits bridge the two DNA Pol modules of Pol2, holding them rigid. This information enabled an atomic model of the leading strand replisome. Interestingly, the model suggests that an OB fold in Dbp2 directs leading ssDNA from CMG to the Pol ε active site. These results complete the DNA path from entry of parental DNA into CMG to exit of daughter DNA from PCNA.

Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks.

The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known. Using reconstituted budding yeast replisomes, we show that mutational inactivation of the leading strand DNA polymerase, Pol ε dNTP depletion, or chemical inhibition of DNA polymerases cause excessive DNA unwinding by the replicative DNA helicase, CMG, demonstrating that budding yeast replisomes lack intrinsic mechanisms that control helicase-polymerase coupling at the fork. Importantly, we find that the Rad53 kinase restricts excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.

Structural studies of the leading strand DNA polymerase in eukaryotes

Structural studies of the leading strand DNA polymerase in eukaryotes

Structural evidence for an essential Fe-S cluster in the catalytic core domain of DNA polymerase ϵ.

DNA polymerase ϵ (Pol ϵ), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2CORE) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2CORE (with the CysX cysteine motif) are likely to coordinate an Fe–S cluster. Here, we present two new crystal structures of Pol2CORE with an Fe–S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol ϵ, Pol ϵ CysAMUT (C2111S/C2133S), and Pol ϵ CysBMUT (C2167S/C2181S) all have an Fe–S cluster that is not present in Pol ϵ CysXMUT (C665S/C668S). Pol ϵ CysAMUT and Pol ϵ CysBMUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysXMUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysXMUT are inviable. In conclusion, Pol ϵ has a single Fe–S cluster bound at the base of the P-domain, and this Fe–S cluster is essential for cell viability and polymerase activity.

DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.