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Graham C Walker - One of the best experts on this subject based on the ideXlab platform.
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rev7 dimerization is important for assembly and function of the REV1 polζ translesion synthesis complex
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Alessandro A Rizzo, Fayemarie Vassel, Nimrat Chatterjee, Sanjay Dsouza, Yunfeng Li, Michael T Hemann, Graham C Walker, Dmitry M. KorzhnevAbstract:The translesion synthesis (TLS) polymerases Polζ and REV1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding REV1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the “safety belt” loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7 −/− cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31 comet . Structurally, the Rev7 dimer can bind only one copy of REV1, revealing an unexpected REV1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the REV1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.
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Rev7 dimerization is important for assembly and function of the REV1/Polζ translesion synthesis complex.
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Alessandro A Rizzo, Fayemarie Vassel, Nimrat Chatterjee, Yunfeng Li, Michael T Hemann, Graham C Walker, Sanjay D'souza, Dmitry M. KorzhnevAbstract:The translesion synthesis (TLS) polymerases Polζ and REV1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding REV1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the “safety belt” loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7 −/− cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31 comet . Structurally, the Rev7 dimer can bind only one copy of REV1, revealing an unexpected REV1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the REV1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.
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identification of small molecule translesion synthesis inhibitors that target the REV1 ct rir protein protein interaction
ACS Chemical Biology, 2017Co-Authors: Vibhavari Sail, Dmitry M. Korzhnev, Alessandro A Rizzo, Nimrat Chatterjee, Graham C Walker, Radha Charan Dash, Zuleyha Ozen, Kyle M HaddenAbstract:Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic REV1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of REV1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase REV1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein–protein interactions (PPIs) between the C-terminal domain of REV1 (REV1-CT) and the REV1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the REV1-CT/RIR PPI is a valid approach for developing a new class of targe...
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Identification of Small Molecule Translesion Synthesis Inhibitors That Target the REV1-CT/RIR Protein−Protein Interaction
ACS Chemical Biology, 2017Co-Authors: Vibhavari Sail, Dmitry M. Korzhnev, Alessandro A Rizzo, Nimrat Chatterjee, Graham C Walker, Radha Charan Dash, Zuleyha Ozen, M. Kyle HaddenAbstract:Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic REV1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of REV1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase REV1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein–protein interactions (PPIs) between the C-terminal domain of REV1 (REV1-CT) and the REV1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the REV1-CT/RIR PPI is a valid approach for developing a new class of targe...
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interaction between the REV1 c terminal domain and the pold3 subunit of polζ suggests a mechanism of polymerase exchange upon REV1 polζ dependent translesion synthesis
Biochemistry, 2016Co-Authors: Yulia Pustovalova, Alessandro A Rizzo, Sanjay Dsouza, Graham C Walker, Mariana T Q Magalhaes, George Korza, Dmitry M. KorzhnevAbstract: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/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι, or Polκ, inserts a nucleotide across a DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (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 Polζ and REV1-interacting regions (RIRs) from Polη, Polι, or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal p...
Todd M Washington - One of the best experts on this subject based on the ideXlab platform.
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the proliferating cell nuclear antigen pcna interacting protein pip motif of dna polymerase η mediates its interaction with the c terminal domain of REV1
Journal of Biological Chemistry, 2016Co-Authors: Elizabeth M Boehm, Kyle T Powers, Christine M Kondratick, Maria Spies, Jon C D Houtman, Todd M WashingtonAbstract:Abstract Y-family DNA polymerases, such as polymerase η, polymerase ι, and polymerase κ, catalyze the bypass of DNA damage during translesion synthesis. These enzymes are recruited to sites of DNA damage by interacting with the essential replication accessory protein proliferating cell nuclear antigen (PCNA) and the scaffold protein REV1. In most Y-family polymerases, these interactions are mediated by one or more conserved PCNA-interacting protein (PIP) motifs that bind in a hydrophobic pocket on the front side of PCNA as well as by conserved REV1-interacting region (RIR) motifs that bind in a hydrophobic pocket on the C-terminal domain of REV1. Yeast polymerase η, a prototypical translesion synthesis polymerase, binds both PCNA and REV1. It possesses a single PIP motif but not an RIR motif. Here we show that the PIP motif of yeast polymerase η mediates its interactions both with PCNA and with REV1. Moreover, the PIP motif of polymerase η binds in the hydrophobic pocket on the REV1 C-terminal domain. We also show that the RIR motif of human polymerase κ and the PIP motif of yeast Msh6 bind both PCNA and REV1. Overall, these findings demonstrate that PIP motifs and RIR motifs have overlapping specificities and can interact with both PCNA and REV1 in structurally similar ways. These findings also suggest that PIP motifs are a more versatile protein interaction motif than previously believed.
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structure and functional analysis of the brct domain of translesion synthesis dna polymerase REV1
Biochemistry, 2013Co-Authors: John M Pryor, Lokesh Gakhar, Todd M WashingtonAbstract:Translesion synthesis (TLS) is a pathway in which specialized, low-fidelity DNA polymerases are used to overcome replication blocks caused by DNA damage. The use of this pathway often results in somatic mutations that can drive carcinogenesis. REV1 is a TLS polymerase found in all eukaryotes that plays a pivotal role in mediating DNA-damage induced mutagenesis. It possesses a BRCA1 C-terminal (BRCT) domain that is required for its function. The REV1-1 allele encodes a mutant form of REV1 with a G193R substitution in this domain, which reduces DNA damage-induced mutagenesis. Despite its clear importance in mutagenic TLS, the role of the BRCT domain is unknown. Here, we report the X-ray crystal structure of the yeast REV1 BRCT domain and show that substitutions in residues constituting its phosphate-binding pocket do not affect mutagenic TLS. This suggests that the role of the REV1 BRCT domain is not to recognize phosphate groups on protein binding partners or on DNA. We also found that the G193 residue is located in a conserved turn region of the BRCT domain, and our in vivo and in vitro studies suggest that the G193R substitution may disrupt REV1 function by destabilizing the fold of the BRCT domain.
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pre steady state kinetic studies show that an abasic site is a cognate lesion for the yeast REV1 protein
DNA Repair, 2011Co-Authors: John M Pryor, Todd M WashingtonAbstract:REV1 is a eukaryotic DNA polymerase that rescues replication forks stalled at sites of DNA damage by inserting nucleotides opposite the damaged template bases. Yeast genetic studies suggest that REV1 plays an important role in rescuing replication forks stalled at one of the most common forms of DNA damage, an abasic site; however, steady state kinetic studies suggest that an abasic site acts as a significant block to nucleotide incorporation by REV1. Here we examined the pre-steady state kinetics of nucleotide incorporation by yeast REV1 with damaged and non-damaged DNA substrates. We found that yeast REV1 is capable of rapid nucleotide incorporation, but only a small fraction of the protein molecules possessed this robust activity. We characterized the nucleotide incorporation by the catalytically robust fraction of yeast REV1 and found that it efficiently incorporated dCTP opposite a template abasic site under pre-steady state conditions. We conclude from these studies that the abasic site is a cognate lesion for REV1.
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pre steady state kinetic studies of protein template directed nucleotide incorporation by the yeast REV1 protein
Biochemistry, 2007Co-Authors: Craig A Howell, Satya Prakash, Todd M WashingtonAbstract:: The yeast REV1 protein (REV1p) is a member of the Y family of DNA polymerases that specifically catalyzes the incorporation of C opposite template G and several types of DNA damage. The X-ray crystal structure of the REV1p-DNA-dCTP ternary complex showed that REV1p utilizes an unusual mechanism of nucleotide incorporation whereby the template residue is displaced from the DNA double helix and the side chain of Arg-324 forms hydrogen bonds with the incoming dCTP. To better understand the impact of this protein-template-directed mechanism on the thermodynamics and kinetics of nucleotide incorporation, we have carried out pre-steady-state kinetic studies with REV1p. Interestingly, we found that REV1p's specificity for incorporating C is achieved solely at the initial nucleotide-binding step, not at the subsequent nucleotide-incorporation step. In this respect, REV1p differs from all previously investigated DNA polymerases. We also found that the base occupying the template position in the DNA impacts nucleotide incorporation more at the nucleotide-binding step than at the nucleotide-incorporation step. These studies provide the first detailed, quantitative information regarding the mechanistic impact of protein-template-directed nucleotide incorporation by REV1p. Moreover, on the basis of these findings and on structures of the unrelated Escherichia coli MutM DNA glycosylase, we suggest the possible structures for the ternary complexes of REV1p with the other incoming dNTPs.
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efficient and error free replication past a minor groove n2 guanine adduct by the sequential action of yeast REV1 and dna polymerase ζ
Molecular and Cellular Biology, 2004Co-Authors: Lajos Haracska, R E Johnson, Todd M Washington, Irina G Minko, Thomas M Harris, Stephen R LloydAbstract:REV1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase ζ (Polζ). REV1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While REV1 plays an indispensable structural role in Polζ-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, REV1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N2-adducted guanines in DNA. To test this idea, we examined whether REV1 could incorporate a C opposite the γ-hydroxy-1,N2-propano-2′deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N2 of guanine. Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that REV1 efficiently incorporates a C opposite this adduct from which Polζ subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the REV1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N2-guanine DNA minor-groove adducts that form in DNA.
Dmitry M. Korzhnev - One of the best experts on this subject based on the ideXlab platform.
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rev7 dimerization is important for assembly and function of the REV1 polζ translesion synthesis complex
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Alessandro A Rizzo, Fayemarie Vassel, Nimrat Chatterjee, Sanjay Dsouza, Yunfeng Li, Michael T Hemann, Graham C Walker, Dmitry M. KorzhnevAbstract:The translesion synthesis (TLS) polymerases Polζ and REV1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding REV1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the “safety belt” loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7 −/− cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31 comet . Structurally, the Rev7 dimer can bind only one copy of REV1, revealing an unexpected REV1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the REV1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.
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Rev7 dimerization is important for assembly and function of the REV1/Polζ translesion synthesis complex.
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Alessandro A Rizzo, Fayemarie Vassel, Nimrat Chatterjee, Yunfeng Li, Michael T Hemann, Graham C Walker, Sanjay D'souza, Dmitry M. KorzhnevAbstract:The translesion synthesis (TLS) polymerases Polζ and REV1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding REV1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the “safety belt” loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7 −/− cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31 comet . Structurally, the Rev7 dimer can bind only one copy of REV1, revealing an unexpected REV1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the REV1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.
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identification of small molecule translesion synthesis inhibitors that target the REV1 ct rir protein protein interaction
ACS Chemical Biology, 2017Co-Authors: Vibhavari Sail, Dmitry M. Korzhnev, Alessandro A Rizzo, Nimrat Chatterjee, Graham C Walker, Radha Charan Dash, Zuleyha Ozen, Kyle M HaddenAbstract:Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic REV1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of REV1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase REV1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein–protein interactions (PPIs) between the C-terminal domain of REV1 (REV1-CT) and the REV1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the REV1-CT/RIR PPI is a valid approach for developing a new class of targe...
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Identification of Small Molecule Translesion Synthesis Inhibitors That Target the REV1-CT/RIR Protein−Protein Interaction
ACS Chemical Biology, 2017Co-Authors: Vibhavari Sail, Dmitry M. Korzhnev, Alessandro A Rizzo, Nimrat Chatterjee, Graham C Walker, Radha Charan Dash, Zuleyha Ozen, M. Kyle HaddenAbstract:Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic REV1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of REV1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase REV1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein–protein interactions (PPIs) between the C-terminal domain of REV1 (REV1-CT) and the REV1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the REV1-CT/RIR PPI is a valid approach for developing a new class of targe...
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interaction between the REV1 c terminal domain and the pold3 subunit of polζ suggests a mechanism of polymerase exchange upon REV1 polζ dependent translesion synthesis
Biochemistry, 2016Co-Authors: Yulia Pustovalova, Alessandro A Rizzo, Sanjay Dsouza, Graham C Walker, Mariana T Q Magalhaes, George Korza, Dmitry M. KorzhnevAbstract: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/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι, or Polκ, inserts a nucleotide across a DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (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 Polζ and REV1-interacting regions (RIRs) from Polη, Polι, or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal p...
Niels De Wind - One of the best experts on this subject based on the ideXlab platform.
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fancd2 and REV1 cooperate in the protection of nascent dna strands in response to replication stress
Nucleic Acids Research, 2015Co-Authors: Yeran Yang, Jacob G Jansen, Fengli Wang, Piya Temviriyanukul, Yingfeng Tu, Lingna Lv, Min Huang, Ting Zhang, Benjamin P C Chen, Niels De WindAbstract:REV1 is a eukaryotic member of the Y-family of DNA polymerases involved in translesion DNA synthesis and genome mutagenesis. Recently, REV1 is also found to function in homologous recombination. However, it remains unclear how REV1 is recruited to the sites where homologous recombination is processed. Here, we report that loss of mammalian REV1 results in a specific defect in replication-associated gene conversion. We found that REV1 is targeted to laser-induced DNA damage stripes in a manner dependent on its ubiquitin-binding motifs, on RAD18, and on monoubiquitinated FANCD2 (FANCD2-mUb) that associates with REV1. Expression of a FANCD2-Ub chimeric protein in RAD18-depleted cells enhances REV1 assembly at laser-damaged sites, suggesting that FANCD2-mUb functions downstream of RAD18 to recruit REV1 to DNA breaks. Consistent with this suggestion we found that REV1 and FANCD2 are epistatic with respect to sensitivity to the double-strand break-inducer camptothecin. REV1 enrichment at DNA damage stripes also partially depends on BRCA1 and BRCA2, components of the FANCD2/BRCA supercomplex. Intriguingly, analogous to FANCD2-mUb and BRCA1/BRCA2, REV1 plays an unexpected role in protecting nascent replication tracts from degradation by stabilizing RAD51 filaments. Collectively these data suggest that REV1 plays multiple roles at stalled replication forks in response to replication stress.
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REV1 is essential in generating g to c transversions downstream of the ung2 pathway but not the msh2 ung2 hybrid pathway
European Journal of Immunology, 2013Co-Authors: Peter H L Krijger, Niels De Wind, Anastasia Tsaalbishtylik, Paul C M Van Den Berk, Heinz JacobsAbstract:Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes are initiated by the enzymatic deamination of cytosine (C) to uracil (U). Uracil-DNA-glycosylase (Ung2) converts uracils into apyrimidinic (AP) sites, which is essential for the generation of transversions (TVs) at G/C basepairs during SHM and for efficient DNA break formation during CSR. Besides Ung2, the mismatch repair protein Msh2 and the translesion synthesis (TLS) DNA polymerase (Pol) REV1 are implicated in SHM and CSR. To further unravel the role of REV1, we studied WT, REV1-deficient, Msh2-deficient, and REV1, Msh2 double-deficient B cells. Loss of REV1 only slightly reduced CSR. During SHM G/C to C/G TVs are generated in both Ung2- and Ung+Msh2-dependent fashions. We found that REV1 is essential for the Msh2-independent generation of these TVs downstream of Ung2-induced AP sites. In the Ung+Msh2 hybrid pathway, REV1 is not essential and can be substituted by an alternative TLS Pol, especially when REV1 is lacking.
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REV1 is essential in generating G to C transversions downstream of the Ung2 pathway but not the Msh2+Ung2 hybrid pathway.
European Journal of Immunology, 2013Co-Authors: Peter H L Krijger, Niels De Wind, Paul C M Van Den Berk, Anastasia Tsaalbi-shtylik, Heinz JacobsAbstract:Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes are initiated by the enzymatic deamination of cytosine (C) to uracil (U). Uracil-DNA-glycosylase (Ung2) converts uracils into apyrimidinic (AP) sites, which is essential for the generation of transversions (TVs) at G/C basepairs during SHM and for efficient DNA break formation during CSR. Besides Ung2, the mismatch repair protein Msh2 and the translesion synthesis (TLS) DNA polymerase (Pol) REV1 are implicated in SHM and CSR. To further unravel the role of REV1, we studied WT, REV1-deficient, Msh2-deficient, and REV1, Msh2 double-deficient B cells. Loss of REV1 only slightly reduced CSR. During SHM G/C to C/G TVs are generated in both Ung2- and Ung+Msh2-dependent fashions. We found that REV1 is essential for the Msh2-independent generation of these TVs downstream of Ung2-induced AP sites. In the Ung+Msh2 hybrid pathway, REV1 is not essential and can be substituted by an alternative TLS Pol, especially when REV1 is lacking.
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the vital role of polymerase ζ and REV1 in mutagenic but not correct dna synthesis across benzo a pyrene dg and recruitment of polymerase ζ by REV1 to replication stalled site
Journal of Biological Chemistry, 2012Co-Authors: Keiji Hashimoto, In Young Yang, Jun Ichi Akagi, Eiji Ohashi, Satoshi Tateishi, Niels De Wind, Fumio Hanaoka, Haruo Ohmori, Masaaki MoriyaAbstract:The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G → T transversions being predominant. Knockout of the REV1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into REV1−/− MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk−/− Polh−/− Poli−/− triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.
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the REV1 translesion synthesis polymerase has multiple distinct dna binding modes
DNA Repair, 2011Co-Authors: Frederik H De Groote, Yuji Masuda, Kenji Kamiya, Niels De Wind, Jacob G Jansen, Dipen M Shah, Gregg SiegalAbstract:Abstract REV1 is a eukaryotic DNA polymerase of the Y family involved in translesion synthesis (TLS), a major damage tolerance pathway that allows DNA replication at damaged templates. Uniquely amongst the Y family polymerases, the N-terminal part of REV1, dubbed the BR CA1 C - t erminal homology (BRCT) region, includes a BRCT domain. While most BRCT domains mediate protein–protein interactions, REV1 contains a predicted α-helix N-terminal to the BRCT domain and in human Replication Factor C (RFC) such a BRCT region endows the protein with DNA binding capacity. Here, we studied the DNA binding properties of yeast and mouse REV1. Our results show that the BRCT region of REV1 specifically binds to a 5′ phosphorylated, recessed, primer–template junction. This DNA binding depends on the extra α-helix, N-terminal to the BRCT domain. Surprisingly, a stretch of 20 amino acids N-terminal to the predicted α-helix is also critical for high-affinity DNA binding. In addition to 5′ primer–template junction binding, REV1 efficiently binds to a recessed 3′ primer–template junction. These dual DNA binding characteristics are discussed in view of the proposed recruitment of REV1 by 5′ primer–template junctions, downstream of stalled replication forks.
Zhigang Wang - One of the best experts on this subject based on the ideXlab platform.
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a non catalytic function of REV1 in translesion dna synthesis and mutagenesis is mediated by its stable interaction with rad5
DNA Repair, 2013Co-Authors: Lisha Kuang, Ying Zhou, Xingang Feng, Lei Wang, Zhigang WangAbstract:Abstract DNA damage tolerance consisting of template switching and translesion synthesis is a major cellular mechanism in response to unrepaired DNA lesions during replication. The REV1 pathway constitutes the major mechanism of translesion synthesis and base damage-induced mutagenesis in model cell systems. REV1 is a dCMP transferase, but additionally plays non-catalytic functions in translesion synthesis. Using the yeast model system, we attempted to gain further insights into the non-catalytic functions of REV1. REV1 stably interacts with Rad5 (a central component of the template switching pathway) via the C-terminal region of REV1 and the N-terminal region of Rad5. Supporting functional significance of this interaction, both the REV1 pathway and Rad5 are required for translesion synthesis and mutagenesis of 1, N 6 -ethenoadenine. Furthermore, disrupting the REV1–Rad5 interaction by mutating REV1 did not affect its dCMP transferase, but led to inactivation of the REV1 non-catalytic function in translesion synthesis of UV-induced DNA damage. Deletion analysis revealed that the C-terminal 21-amino acid sequence of REV1 is uniquely required for its interaction with Rad5 and is essential for its non-catalytic function. Deletion analysis additionally implicated a C-terminal region of REV1 in its negative regulation. These results show that a non-catalytic function of REV1 in translesion synthesis and mutagenesis is mediated by its interaction with Rad5.
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Abstract 2553: A non-catalytic function of REV1 in translesion DNA synthesis and mutagenesis is mediated by its stable interaction with Rad5
Cancer Research, 2012Co-Authors: Lisha Kuang, Ying Zhou, Zhigang WangAbstract:DNA damage tolerance is a major cellular mechanism in response to unrepaired DNA lesions during replication. It allows replication to completion in the presence of DNA lesions that block replicative polymerases. Damage tolerance consists of two major mechanisms: template switching and translesion DNA synthesis. Translesion synthesis directly copies damaged sites of the DNA template during replication. Consequently, error-prone translesion synthesis constitutes the major mechanism of base damage-induced mutagenesis. In eukaryotes, translesion synthesis is carried out by the REV1-Polα pathway. REV1 possesses a dCMP transferase activity, and is a member of the Y family of DNA polymerases. Additionally, it is widely believed that REV1 plays non-catalytic functions in translesion synthesis. However, the non-catalytic function of REV1 is not clearly defined. Using the yeast model system, we have investigated non-catalytic functions of REV1 in translesion synthesis and base damage-induced mutagenesis. Specifically, we examined the role of REV1-Rad5 interaction in translesion synthesis of UV damage and a site-specific 1, N 6 -ethenoadenine (a DNA lesion of lipid peroxidation products). Although Rad5 is better known for its role in template switching, it stably interacts with REV1. Interaction domains in the REV1 and Rad5 proteins were defined. Translesion synthesis and mutagenesis required Rad5 in cells. Disrupting the REV1-Rad5 interaction by mutating REV1 did not affect the dCMP transferase of REV1, but led to inactivation of the translesion synthesis pathway. These results show that Rad5 is an indispensible component of the REV1-Pol ≤ pathway for translesion synthesis and base damage-induced mutagenesis, and a non-catalytic function of REV1 in translesion DNA synthesis is mediated by its stable interaction with Rad5. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2553. doi:1538-7445.AM2012-2553
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the catalytic function of the REV1 dcmp transferase is required in a lesion specific manner for translesion synthesis and base damage induced mutagenesis
Nucleic Acids Research, 2010Co-Authors: Ying Zhou, Jillian Wang, Yanbin Zhang, Zhigang WangAbstract:The REV1-Polf pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes. While it is widely believed that REV1 plays a non-catalytic function in translesion synthesis, the role of its dCMP transferase activity remains uncertain. To determine the relevance of its catalytic function in translesion synthesis, we separated the REV1 dCMP transferase activity from its non-catalytic function in yeast. This was achieved by mutating two conserved amino acid residues in the catalytic domain of REV1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact. In this mutant strain, whereas translesion synthesis and mutagenesis of UV radiation were fully functional, those of a site-specific 1,N 6 -ethenoadenine were severely deficient. Specifically, the predominant A!G mutations resulting from C insertion opposite the lesion were abolished. Therefore, translesion synthesis and mutagenesis of 1,N 6 -ethenoadenine require the catalytic function of the REV1 dCMP transferase, in contrast to those of UV lesions, which only require the non-catalytic function of REV1. These results show that the catalytic function of the REV1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.
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Translesion synthesis of acetylaminofluorene‐dG adducts by DNA polymerase ζ is stimulated by yeast REV1 protein
Nucleic Acids Research, 2004Co-Authors: Huiyun Shen, Bo Zhao, Zhigang WangAbstract:Translesion synthesis is an important mechanism in response to unrepaired DNA lesions during replication. The DNA polymerase z (Polz) mutagenesis pathway is a major error-prone translesion synthesis mechanism requiring Polz and REV1. In addition to its dCMP transferase, a non-catalytic function of REV1 is suspected in cellular response to certain types of DNA lesions. However, it is not well understood about the non-catalytic function of REV1 in translesion synthesis. We have analyzed the role of REV1 in translesion synthesis of an acetylaminofluorene (AAF)-dG DNA adduct. Purified yeast REV1 was essentially unresponsive to a template AAF-dG DNA adduct, in contrast to its efficient C insertion opposite a template 1,N 6 -ethenoadenine adduct. Purified yeast Polz was very inefficient in the bypass of the AAF-dG adduct. Combining REV1 and Polz, however, led to a synergistic effect on translesion synthesis. REV1 protein enhanced Polz-catalyzed nucleotide insertion opposite the AAF-dG adduct and strongly stimulated Polz-catalyzed extension from opposite the lesion. REV1 also stimulated the deficient synthesis by Polz at the very end of undamaged DNA templates. Deleting the C-terminal 205 aa of REV1 did not affect its dCMP transferase activity, but abolished its stimulatory activity on Polz-catalyzed extension from opposite the AAF-dG adduct. These results suggest that translesion synthesis of AAF-dG adducts by Polz is stimulated by REV1 protein in yeast. Consistent with the in vitro results, both Polz and REV1 were found to be equally important for error-prone translesion synthesis across from AAF-dG DNA adducts in yeast cells.
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translesion synthesis of acetylaminofluorene dg adducts by dna polymerase ζ is stimulated by yeast REV1 protein
Nucleic Acids Research, 2004Co-Authors: Huiyun Shen, Bo Zhao, Zhigang WangAbstract:Translesion synthesis is an important mechanism in response to unrepaired DNA lesions during replication. The DNA polymerase z (Polz) mutagenesis pathway is a major error-prone translesion synthesis mechanism requiring Polz and REV1. In addition to its dCMP transferase, a non-catalytic function of REV1 is suspected in cellular response to certain types of DNA lesions. However, it is not well understood about the non-catalytic function of REV1 in translesion synthesis. We have analyzed the role of REV1 in translesion synthesis of an acetylaminofluorene (AAF)-dG DNA adduct. Purified yeast REV1 was essentially unresponsive to a template AAF-dG DNA adduct, in contrast to its efficient C insertion opposite a template 1,N 6 -ethenoadenine adduct. Purified yeast Polz was very inefficient in the bypass of the AAF-dG adduct. Combining REV1 and Polz, however, led to a synergistic effect on translesion synthesis. REV1 protein enhanced Polz-catalyzed nucleotide insertion opposite the AAF-dG adduct and strongly stimulated Polz-catalyzed extension from opposite the lesion. REV1 also stimulated the deficient synthesis by Polz at the very end of undamaged DNA templates. Deleting the C-terminal 205 aa of REV1 did not affect its dCMP transferase activity, but abolished its stimulatory activity on Polz-catalyzed extension from opposite the AAF-dG adduct. These results suggest that translesion synthesis of AAF-dG adducts by Polz is stimulated by REV1 protein in yeast. Consistent with the in vitro results, both Polz and REV1 were found to be equally important for error-prone translesion synthesis across from AAF-dG DNA adducts in yeast cells.
