Look Ma, no PCNA: how DNA polymerase ε synthesizes long stretches of DNA without a processivity factor.
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ABSTRACT:
SUBMITTER: Zahn KE
PROVIDER: S-EPMC4226222 | biostudies-literature | 2014 Jan
REPOSITORIES: biostudies-literature
Publications
Look Ma, no PCNA: how DNA polymerase ε synthesizes long stretches of DNA without a processivity factor.
Zahn Karl E KE Doublié Sylvie S
Nature structural & molecular biology 20140101 1
Similar Datasets
Project description:Family B DNA polymerases comprise polymerase and 3' ->5' exonuclease domains, and detect a mismatch in a newly synthesized strand to remove it in cooperation with Proliferating cell nuclear antigen (PCNA), which encircles the DNA to provide a molecular platform for efficient protein-protein and protein-DNA interactions during DNA replication and repair. Once the repair is completed, the enzyme must stop the exonucleolytic process and switch to the polymerase mode. However, the cue to stop the degradation is unclear. We constructed several PCNA mutants and found that the exonuclease reaction was enhanced in the mutants lacking the conserved basic patch, located on the inside surface of PCNA. These mutants may mimic the Pol/PCNA complex processing the mismatched DNA, in which PCNA cannot interact rigidly with the irregularly distributed phosphate groups outside the dsDNA. Indeed, the exonuclease reaction with the wild type PCNA was facilitated by mismatched DNA substrates. PCNA may suppress the exonuclease reaction after the removal of the mismatched nucleotide. PCNA seems to act as a "brake" that stops the exonuclease mode of the DNA polymerase after the removal of a mismatched nucleotide from the substrate DNA, for the prompt switch to the DNA polymerase mode.
| S-EPMC5353730 | biostudies-literature
Project description:The papillomavirus (PV) helicase protein E1 recruits components of the cellular DNA replication machinery to the PV replication fork, such as Replication Protein A (RPA), DNA polymerase α-primase (pol α) and topoisomerase I (topo I). Here we show that E1 binds to DNA polymerase ϵ (pol ϵ) and dramatically stimulates the DNA synthesis activity of pol ϵ. This stimulation of pol ϵ by E1 is highly specific and occurs even in the absence of the known pol ϵ cofactors Replication Factor C (RFC), Proliferating Cell Nuclear Antigen (PCNA) and RPA. This stimulation is due to an increase in the processivity of pol ϵ and occurs independently of pol ϵ's replication cofactors. This increase in processivity is dependent on the ability of the E1 helicase to hydrolyze ATP, suggesting it is dependent on E1's helicase action. In addition, RPA, thought to be vital for processive DNA synthesis by both pol ϵ and pol δ, was found to be dispensable for processive synthesis by pol ϵ in the presence of E1. Overall, E1 appears to be conferring processivity to pol ϵ by directly tethering pol ϵ to the DNA parental strand and towing ϵ behind the E1 helicase as the replication fork progresses; and thereby apparently obviating the need for RPA for leading strand synthesis. Thus far only pol α and pol δ have been implicated in the DNA replication of mammalian viruses; this is the first reported example of a virus recruiting pol ϵ. Furthermore, this demonstrates a unique capacity of a viral helicase having evolved to stimulate a cellular replicative DNA polymerase.
| S-EPMC5758917 | biostudies-literature
Project description:The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase ε (Polε). We observed that, under near-physiological conditions, CTF18-RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Polε. During efficient PCNA loading, CTF18-RFC and Polε assembled at a 3΄ primer-template junction cooperatively, and directed PCNA to the loading site. Site-specific photo-crosslinking of directly interacting proteins at the primer-template junction showed similar cooperative binding, in which the catalytic N-terminal portion of Polε acted as the major docking protein. In the PCNA-loading intermediate with ATPγS, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Polε placed in DNA synthesis mode using a substrate DNA with a deoxidised 3΄ primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3΄ primer-template junction. Furthermore, PCNA and CTF18-RFC-Polε complex engaged in stable trimeric assembly on the template DNA and synthesised DNA efficiently. Thus, CTF18-RFC appears to be involved in leading-strand DNA synthesis through its interaction with Polε, and can load PCNA onto DNA when Polε is not in DNA synthesis mode to restore DNA synthesis.
| S-EPMC5416766 | biostudies-literature
Project description:Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol δ replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol δ from cells reduces BIR, whereas depletion of Pol ε has no effect.
| S-EPMC6862718 | biostudies-literature
Project description:During eukaryotic replication, DNA polymerases ε (Polε) and δ (Polδ) synthesize the leading and lagging strands, respectively. In a long-known contradiction to this model, defects in the fidelity of Polε have a much weaker impact on mutagenesis than analogous Polδ defects. It has been previously proposed that Polδ contributes more to mutation avoidance because it proofreads mismatches created by Polε in addition to its own errors. However, direct evidence for this model was missing. We show that, in yeast, the mutation rate increases synergistically when a Polε nucleotide selectivity defect is combined with a Polδ proofreading defect, demonstrating extrinsic proofreading of Polε errors by Polδ. In contrast, combining Polδ nucleotide selectivity and Polε proofreading defects produces no synergy, indicating that Polε cannot correct errors made by Polδ. We further show that Polδ can remove errors made by exonuclease-deficient Polε in vitro. These findings illustrate the complexity of the one-strand-one-polymerase model where synthesis appears to be largely divided, but Polδ proofreading operates on both strands.
| S-EPMC7084062 | biostudies-literature
Project description:Vaccinia virus is the prototypic poxvirus. The 192 kilobase double-stranded DNA viral genome encodes most if not all of the viral replication machinery. The vaccinia virus DNA polymerase is encoded by the E9L gene. Sequence analysis indicates that E9 is a member of the B family of replicative polymerases. The enzyme has both polymerase and 3'-5' exonuclease activities, both of which are essential to support viral replication. Genetic analysis of E9 has identified residues and motifs whose alteration can confer temperature-sensitivity, drug resistance (phosphonoacetic acid, aphidicolin, cytosine arabinsode, cidofovir) or altered fidelity. The polymerase is involved both in DNA replication and in recombination. Although inherently distributive, E9 gains processivity by interacting in a 1:1 stoichiometry with a heterodimer of the A20 and D4 proteins. A20 binds to both E9 and D4 and serves as a bridge within the holoenzyme. The A20/D4 heterodimer has been purified and can confer processivity on purified E9. The interaction of A20 with D4 is mediated by the N'-terminus of A20. The D4 protein is an enzymatically active uracil DNA glycosylase. The DNA-scanning activity of D4 is proposed to keep the holoenzyme tethered to the DNA template but allow polymerase translocation. The crystal structure of D4, alone and in complex with A201-50 and/or DNA has been solved. Screens for low molecular weight compounds that interrupt the A201-50/D4 interface have yielded hits that disrupt processive DNA synthesis in vitro and/or inhibit plaque formation. The observation that an active DNA repair enzyme is an integral part of the holoenzyme suggests that DNA replication and repair may be coupled.
| S-EPMC5476500 | biostudies-literature
Project description:The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n-2 and n-4/n-5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3'-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases.
| S-EPMC4333401 | biostudies-literature
Project description: Not available
| S-EPMC3694787 | biostudies-literature
Project description:Kaposi's Sarcoma Herpesvirus (KSHV) is the causative agent of several human diseases. There are few effective treatments available to treat infection and KSHV oncogenesis. Disrupting the KSHV infectious cycle would diminish the viral spread. The KSHV lytic phase and production of new virions require efficient copying and packaging of the KSHV genome. KSHV encodes its own lytic DNA replication machinery, including the processivity factor (PF-8), which presents itself as an attractive target for antiviral development. We characterized PF-8 at the single molecule level using transmission electron microscopy to identify key molecular interactions that mediate viral DNA replication initiation. Our results indicate that PF-8 forms oligomeric ring structures (tetramer, hexamer, and/or dodecamer) similar to the related Epstein-Barr virus processivity factor (BMRF1). Our DNA positional mapping revealed high-frequency binding locations of PF-8 within the lytic origin of replication (OriLyt). A multi-variable analysis of PF-8 DNA-binding activity with three mutant OriLyts provides new insights into the mechanisms that PF-8 associates with viral DNA and complexes to form multi-ring-like structures. Collectively, these data enhance the mechanistic understanding of the molecular interactions (protein-protein and protein-DNA) of an essential KSHV DNA replication protein.
| S-EPMC11860742 | biostudies-literature
Project description:Translesion synthesis (TLS), the process by which DNA polymerases replicate through DNA lesions, is the source of most DNA damage-induced mutations. Sometimes TLS is carried out by replicative polymerases that have evolved to synthesize DNA on non-damaged templates. Most of the time, however, TLS is carried out by specialized translesion polymerases that have evolved to synthesize DNA on damaged templates. TLS requires the mono-ubiquitylation of the replication accessory factor proliferating cell nuclear antigen (PCNA). PCNA and ubiquitin-modified PCNA (UbPCNA) stimulate TLS by replicative and translesion polymerases. Two mutant forms of PCNA, one with an E113G substitution and one with a G178S substitution, support normal cell growth but inhibit TLS thereby reducing mutagenesis in yeast. A re-examination of the structures of both mutant PCNA proteins revealed substantial disruptions of the subunit interface that forms the PCNA trimer. Both mutant proteins have reduced trimer stability with the G178S substitution causing a more severe defect. The mutant forms of PCNA and UbPCNA do not stimulate TLS of an abasic site by either replicative Pol δ or translesion Pol η. Normal replication by Pol η was also impacted, but normal replication by Pol δ was much less affected. These findings support a model in which reduced trimer stability causes these mutant PCNA proteins to occasionally undergo conformational changes that compromise their ability to stimulate TLS by both replicative and translesion polymerases.
| S-EPMC3636165 | biostudies-literature