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Crystallization and X-ray diffraction analysis of the ternary complex of the C-terminal domain of human REV1 in complex with REV7 bound to a REV3 fragment involved in translesion DNA synthesis.

ABSTRACT: REV1, REV3 and REV7 are pivotal proteins in translesion DNA synthesis that allows DNA synthesis to continue even in the presence of DNA damage. REV1 and REV3 are error-prone DNA polymerases, while REV7 acts as an adaptor protein that links them together. A ternary complex of the C-terminal domain of human REV1 in complex with REV7 bound to a REV3 fragment has been crystallized. The crystals belonged to space group P3(1)21, with unit-cell parameters a = b = 74.7, c = 124.5?Å.

SUBMITTER: Kikuchi S

PROVIDER: S-EPMC3412784 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Crystallization and X-ray diffraction analysis of the ternary complex of the C-terminal domain of human REV1 in complex with REV7 bound to a REV3 fragment involved in translesion DNA synthesis.

Kikuchi Sotaro S Hara Kodai K Shimizu Toshiyuki T Sato Mamoru M Hashimoto Hiroshi H

Acta crystallographica. Section F, Structural biology and crystallization communications 20120727 Pt 8

REV1, REV3 and REV7 are pivotal proteins in translesion DNA synthesis that allows DNA synthesis to continue even in the presence of DNA damage. REV1 and REV3 are error-prone DNA polymerases, while REV7 acts as an adaptor protein that links them together. A ternary complex of the C-terminal domain of human REV1 in complex with REV7 bound to a REV3 fragment has been crystallized. The crystals belonged to space group P3(1)21, with unit-cell parameters a = b = 74.7, c = 124.5 Å. ...[more]

PMID: 22869133

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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.

Project description:DNA polymerase zeta (Polzeta) is an error-prone DNA polymerase involved in translesion DNA synthesis. Polzeta consists of two subunits: the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847-1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Polzeta to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our results will provide a general structural basis for understanding the REV7 interaction.

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Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation.

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Rev7 dimerization is important for assembly and function of the Rev1/Polζ translesion synthesis complex.

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Structure of the human Rev1-DNA-dNTP ternary complex.

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

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 (?, ?, ?, or Rev1) inserting a nucleotide opposite the lesion and with the heterodimeric B-family polymerase ?, 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 ?, ?, and ? and with the Rev7 subunit of polymerase ?. We report the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol ? complex, and the Pol ? 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.

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Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362.

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Caffeine abolishes the ultraviolet-induced REV3 translesion replication pathway in mouse cells.

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Multifaceted recognition of vertebrate Rev1 by translesion polymerases ? and ?.

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| S-EPMC3406723 | biostudies-literature

Crystallization and diffraction of an Isl1-Ldb1 complex.

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| S-EPMC3515390 | biostudies-literature

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