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The C-terminal domain of human Rev1 contains independent binding sites for DNA polymerase ? and Rev7 subunit of polymerase ?.

ABSTRACT: Human Rev1 is a translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap-filling. Rev1 plays an essential structural role in TLS by providing a binding platform for other TLS polymerases that insert nucleotides across DNA lesions (pol?, pol?, pol?) and extend the distorted primer-terminus (pol?). We use NMR spectroscopy to demonstrate that the Rev1 C-terminal domain utilizes independent interaction interfaces to simultaneously bind a fragment of the 'inserter' pol? and Rev7 subunit of the 'extender' pol?, thereby serving as a cassette that may accommodate several polymerases making them instantaneously available for TLS.

SUBMITTER: Pustovalova Y

PROVIDER: S-EPMC3572780 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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The C-terminal domain of human Rev1 contains independent binding sites for DNA polymerase η and Rev7 subunit of polymerase ζ.

Pustovalova Yulia Y Bezsonova Irina I Korzhnev Dmitry M DM

FEBS letters 20120722 19

Human Rev1 is a translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap-filling. Rev1 plays an essential structural role in TLS by providing a binding platform for other TLS polymerases that insert nucleotides across DNA lesions (polη, polι, polκ) and extend the distorted primer-terminus (polς). We use NMR spectroscopy to demonstrate that the Rev1 C-terminal domain utilizes independent interaction interfaces to simultaneously ...[more]

PMID: 22828282

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Structural basis of recruitment of DNA polymerase ζ by interaction between REV1 and REV7 proteins.

Project description:REV1, REV3, and REV7 are pivotal proteins in translesion DNA synthesis, which allows DNA synthesis even in the presence of DNA damage. REV1 and REV3 are error-prone DNA polymerases and function as inserter and extender polymerases in this process, respectively. REV7 interacts with both REV1 and REV3, acting as an adaptor that functionally links the two, although the structural basis of this collaboration remains unclear. Here, we show the crystal structure of the ternary complex, composed of the C-terminal domain of human REV1, REV7, and a REV3 fragment. The REV1 C-terminal domain adopts a four-helix bundle that interacts with REV7. A linker region between helices 2 and 3, which is conserved among mammals, interacts with the β-sheet of REV7. Remarkably, the REV7-binding interface is distinct from the binding site of DNA polymerase η or κ. Thus, the REV1 C-terminal domain might facilitate polymerase switching by providing a scaffold for both inserter and extender polymerases to bind. Our structure reveals the basis of DNA polymerase ζ (a complex of REV3 and REV7) recruitment to the stalled replication fork and provides insight into the mechanism of polymerase switching.

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Yeast Rev1 protein promotes complex formation of DNA polymerase zeta with Pol32 subunit of DNA polymerase delta.

Project description:Yeast DNA polymerase (Pol) delta, essential for DNA replication, is comprised of 3 subunits, Pol3, Pol31, and Pol32. Of these, the catalytic subunit Pol3 and the second subunit Pol31 are essential, whereas the Pol32 subunit is not essential for DNA replication. Although Pol32 is an integral component of Pol delta, it is also required for translesion synthesis (TLS) by Pol zeta. To begin to decipher the bases of Pol32 involvement in Pol zeta-mediated TLS, here we examine whether Pol32 physically interacts with Pol zeta or its associated proteins and provide evidence for the physical interaction of Pol32 with Rev1. Rev1 plays an indispensable structural role in Pol zeta-mediated TLS and it binds the Rev3 catalytic subunit of Pol zeta. Here, we show that although Pol32 does not directly bind Pol zeta, Pol32 can bind the Rev1-Pol zeta complex through its interaction with Rev1. We find that Pol32 binding has no stimulatory effect on DNA synthesis either by Rev1 in the Rev1-Pol32 complex or by Pol zeta in the Pol zeta-Rev1-Pol32 complex, irrespective of whether proliferating cell nuclear antigen has been loaded onto DNA or not. We discuss evidence for the biological significance of Rev1 binding to Pol32 for Pol zeta function in TLS and suggest a structural role for Rev1 in modulating the binding of Pol zeta with Pol32 in Pol delta stalled at a lesion site.

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REV1 and polymerase ζ facilitate homologous recombination repair.

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Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ.

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Crystal structure of human REV7 in complex with a human REV3 fragment and structural implication of the interaction between DNA polymerase zeta and REV1.

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NMR structure and dynamics of the C-terminal domain from human Rev1 and its complex with Rev1 interacting region of DNA polymerase η.

Project description:Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ, and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases.

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Purification of Drosophila DNA polymerase zeta by REV1 protein-affinity chromatography.

Project description:Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol zeta (DNA polymerase zeta) from Drosophila melanogaster (Dmpol zeta). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol zeta was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2',3'-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)(10:1) as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol zeta showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol zeta were not detected. Drosophila REV7 interacted with Dmpol zeta in vitro, but did not influence the DNA synthesis activity of Dmpol zeta. The present study is the first report about characterization of purified pol zeta from multicellular organisms, and the second concerning the characterization of yeast pol zeta.

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Bypass of DNA interstrand crosslinks by a Rev1-DNA polymerase ζ complex.

Project description:DNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based interstrand crosslink (ICL) with an 8-atom linker between the crosslinked bases. The Rev1-Pol ζ complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1-Pol ζ. Rev1-Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.

| S-EPMC7470978 | biostudies-literature

Structural basis of Rev1-mediated assembly of a quaternary vertebrate translesion polymerase complex consisting of Rev1, heterodimeric polymerase (Pol) ζ, and Pol κ.

Project description:DNA synthesis across lesions during genomic replication requires concerted actions of specialized DNA polymerases in a potentially mutagenic process known as translesion synthesis. Current models suggest that translesion synthesis in mammalian cells is achieved in two sequential steps, with a Y-family DNA polymerase (κ, η, ι, or Rev1) inserting a nucleotide opposite the lesion and with the heterodimeric B-family polymerase ζ, consisting of the catalytic Rev3 subunit and the accessory Rev7 subunit, replacing the insertion polymerase to carry out primer extension past the lesion. Effective translesion synthesis in vertebrates requires the scaffolding function of the C-terminal domain (CTD) of Rev1 that interacts with the Rev1-interacting region of polymerases κ, η, and ι and with the Rev7 subunit of polymerase ζ. We report the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol ζ complex, and the Pol κ Rev1-interacting region. Yeast two-hybrid assays were employed to identify important interface residues of the translesion polymerase complex. The structural elucidation of such a quaternary translesion polymerase complex encompassing both insertion and extension polymerases bridged by the Rev1 CTD provides the first molecular explanation of the essential scaffolding function of Rev1 and highlights the Rev1 CTD as a promising target for developing novel cancer therapeutics to suppress translesion synthesis. Our studies support the notion that vertebrate insertion and extension polymerases could structurally cooperate within a megatranslesion polymerase complex (translesionsome) nucleated by Rev1 to achieve efficient lesion bypass without incurring an additional switching mechanism.

| S-EPMC3460478 | biostudies-literature

Interaction between the Rev1 C-Terminal Domain and the PolD3 Subunit of Pol? Suggests a Mechanism of Polymerase Exchange upon Rev1/Pol?-Dependent Translesion Synthesis.

Project description:Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Pol?-dependent TLS. In the first step, a Y-family TLS enzyme, typically Pol?, Pol?, or Pol?, inserts a nucleotide across a DNA lesion. In the second step, a four-subunit B-family DNA polymerase Pol? (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Pol? and Rev1-interacting regions (RIRs) from Pol?, Pol?, or Pol?. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Pol? whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of "inserter" to "extender" DNA polymerase switch upon Rev1/Pol?-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the "inserter" Pol?, Pol?, or Pol? from its complex with Rev1, and (ii) facilitates assembly of the four-subunit "extender" Pol? through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits.

| S-EPMC4898654 | biostudies-literature

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