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    Michael R Volkert PhD

    TitleProfessor
    InstitutionUniversity of Massachusetts Medical School
    DepartmentMicrobiology and Physiological Systems
    AddressUniversity of Massachusetts Medical School
    55 Lake Avenue North
    Worcester MA 01655
    Phone508-856-2314
      Other Positions
      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentMolecular Genetics and Microbiology

      InstitutionUMMS - Programs, Centers and Institutes
      DepartmentBacterial Genetics and Pathogenesis

        Overview 
        Narrative

        Academic Background

        Ph. D. (1977) Rutgers University

        DNA Repair and Damage Prevention Genes

        Photo: Michael 
R. VolkertDNA repair and damage prevention genes function to maintain the integrity of the genome by preventing mutagenesis and lethality in response to DNA damage produced by endogenous and exogenous agents. The repair genes act, either by repairing damaged bases, restoring them to their undamaged state, or by removing damaged bases from DNA and replacing them. The protection genes function either by detoxifying mutagenic DNA damaging agents, or by protecting DNA from interaction with such agents. DNA repair deficiencies in humans result in an increased incidence of cancer in affected individuals, underscoring the importance of developing a thorough understanding of human DNA repair and protection genes and their mechanisms of action.

        Identification and characterization of human oxidative repair and protection genes. We are using functional genomics to identify human DNA repair genes. The basic methods we use are to introduce and express human cDNAs in E. coli mutants defective in repair of oxidative DNA damage. The inability of these E. coli mutants to repair oxidative DNA damage causes a mutator phenotype that results from spontaneous oxidative DNA damage. Expression of human DNA repair genes, or genes that prevent oxidative DNA damage complement the mutator phenotype and are easily identified by their colony phenotype. The genes identified by these procedures are then analyzed using biochemical and genetic approaches. The methods include the use of bacterial, yeast and mammalian genetics and molecular biology techniques in order to determine the activities of the gene products, their DNA sequences, and the biochemical processes that allow interspecies phenotypic complementation to occur.

        Our initial searches resulted in the identification of the human OXR1 and PC4 genes as two genes that protect eukaryotes from oxidative mutagenesis. These two genes are able to complement repair deficient mutants ofE. coli and suppress the mutator phenotype. We have made mutants of yeast genes homologous to OXR1 and PC4 and demonstrated that these mutants are sensitive to treatments with the oxidative agent hydrogen peroxide. We are now in the process of determining the mechanisms by which these genes protect cells from the consequences of oxidative damage and are examining their roles in oxidation protection in mammalian cells. We are also continuing to search for more human genes that are able to complement the mutator phenotype of the oxidation sensitive strains of E. coli in order to expand our collection of this class of genes.

        OXR1 localizes to mitochondria and is induced in response to oxidative stress in yeast and in human cells. Mitochondria produce reactive oxygen species as a by-product of energy production, thus localization of gene products that protect cells from oxidative damage may be related to its cellular function and current research efforts focus on this aspect of OXR1.

        PC4 interacts with the human XPG protein in vitro, causing its displacement from DNA. XPG is a key player in several types of DNA repair. Our results suggest that PC4 functions in an XPG-dependent pathway of DNA repair specific for oxidative DNA damage and our current research focuses on this possibility.



        Rotation Projects

        Rotation Projects

        Construction and testing of OXR1 inhibitory RNAs. The OXR1 gene is induced by oxidative stress and localizes to mitochondria in human and yeast cells. In yeast we have shown that OXR1 is required for resistance to treatments with the oxidative DNA damaging agent hydrogen peroxide and that resistance can be restored to the yeast mutant strain by expression of human OXR1. This result suggests that OXR1 function is conserved from yeast to human forms and further suggests a role for human OXR1 in resistance to oxidative stress. We will now test this directly by expressing short inhibitory RNAs (siRNA) in human cells and testing for oxidation sensitivity. To accomplish this, we have identified and produced a set of siRNAs. These siRNA expressing oligonucleotides now need to be cloned into the appropriate expression vectors and introduced into human HeLa cells. The cells then need to be tested for OXR1 expression by western blotting methods to screen for inhibition using the anti OXR1 antibody. If inhibition is detected, then the cells will be tested for resistance to hydrogen peroxide and other DNA oxidizing agents.

        Genetic analysis of the human OXR2 gene. Higher eukaryotes have more than one OXR gene, whereas lower eukaryotes such as yeast have only one. The human OXR2 gene is a paralog of the human OXR1 gene discovered in our recent search for DNA protection genes. This gene is about 50% identical to human OXR1 and about 65% similar. OXR2 has been cloned. A rotation project will involve the comparison of this gene with OXR1 in terms of its ability to complement the mutator phenotype of a bacterial mutM mutY strain and to assess its ability to complement the oxidation sensitivity of the yeast OXR deletion mutant strain.

        Analysis of dominant negative alleles of human PC4. Yeast cells lacking their PC4 ortholog Sub1are sensitive to treatments with the oxidative agent hydrogen peroxide. Since human PC4 is able to protect bacterial cells from mutagenesis by oxidative agents and is able to complement the peroxide sensitivity of a yeast sub1 deletion mutant strain, PC4 function appears to be conserved from the yeast to the human forms. This role for PC4 requires its DNA binding activity and our in vitro studies indicate that human PC4 functions in DNA repair. Therefore human PC4 is likely to function in repair of oxidative DNA damage in mammalian cells. Since PC4 appears to be a stable protein, it may be a poor candidate for siRNA inhibition methods. Therefore we will test several potential dominant negative alleles of human PC4 for their ability to inhibit the function of the wild type allele. PC4 works as a dimer to bind to unpaired regions of double-stranded DNA. We have several mutant alleles of PC4 that were produced by site-directed mutagenesis, that specifically inactivate PC4’s DNA binding activity. These mutant alleles should form heterodimers with the wild type PC4 protein and their over-expression should result in a predominance of dimers containing one mutant subunit. Since binding to DNA by dimers is generally cooperative, the binding affinity of a dimer is drastically reduced by the presence of a defective subunit. Thus. Over-expression of a mutant PC4 allele should inactivate or severely impair the DNA binding required for its oxidation resistance. We will test the ability of the mutant alleles to inhibit PC4 function by co-expressing mutant and wild type alleles in yeast and testing for loss of complementation of peroxide resistance in the sub1 deletion mutant, and/or by co-expression in bacteria and testing for loss of the PC4 antimutator function seen in the mutM mutY strain of E. coli. If PC4 inactivation by the mutant PC4 alleles is seen in these tests, then the mutant alleles of PC4 will be expressed in HeLa cells and the transfected cells tested for loss of peroxide resistance.



        Bibliographic 
        selected publications
        List All   |   Timeline
        1. Yu L, Volkert MR. Differential Requirement for SUB1 in Chromosomal and Plasmid Double-Strand DNA Break Repair. PLoS One. 2013; 8(3):e58015.
          View in: PubMed
        2. Yu L, Volkert MR. UV damage regulates alternative polyadenylation of the RPB2 gene in yeast. Nucleic Acids Res. 2013 Mar 1; 41(5):3104-14.
          View in: PubMed
        3. Murphy KC, Volkert MR. Structural/functional analysis of the human OXR1 protein: identification of exon 8 as the anti-oxidant encoding function. BMC Mol Biol. 2012; 13(1):26.
          View in: PubMed
        4. Rippa V, Duilio A, di Pasquale P, Amoresano A, Landini P, Volkert MR. Preferential DNA damage prevention by the E. coli AidB gene: A new mechanism for the protection of specific genes. DNA Repair (Amst). 2011 Sep 5; 10(9):934-41.
          View in: PubMed
        5. Rippa V, Amoresano A, Esposito C, Landini P, Volkert M, Duilio A. Specific DNA binding and regulation of its own expression by the AidB protein in Escherichia coli. J Bacteriol. 2010 Dec; 192(23):6136-42.
          View in: PubMed
        6. Volkert MR, Wang JY, Elliott NA. A functional genomics approach to identify and characterize oxidation resistance genes. Methods Mol Biol. 2008; 477:331-42.
          View in: PubMed
        7. Durand M, Kolpak A, Farrell T, Elliott NA, Shao W, Brown M, Volkert MR. The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins. BMC Cell Biol. 2007; 8:13.
          View in: PubMed
        8. Matijasevic Z, Volkert MR. Base excision repair sensitizes cells to sulfur mustard and chloroethyl ethyl sulfide. DNA Repair (Amst). 2007 Jun 1; 6(6):733-41.
          View in: PubMed
        9. Liang X, Pickering MT, Cho NH, Chang H, Volkert MR, Kowalik TF, Jung JU. Deregulation of DNA damage signal transduction by herpesvirus latency-associated M2. J Virol. 2006 Jun; 80(12):5862-74.
          View in: PubMed
        10. Wang JY, Sarker AH, Cooper PK, Volkert MR. The single-strand DNA binding activity of human PC4 prevents mutagenesis and killing by oxidative DNA damage. Mol Cell Biol. 2004 Jul; 24(13):6084-93.
          View in: PubMed
        11. Elliott NA, Volkert MR. Stress induction and mitochondrial localization of Oxr1 proteins in yeast and humans. Mol Cell Biol. 2004 Apr; 24(8):3180-7.
          View in: PubMed
        12. Wyrzykowski J, Volkert MR. The Escherichia coli methyl-directed mismatch repair system repairs base pairs containing oxidative lesions. J Bacteriol. 2003 Mar; 185(5):1701-4.
          View in: PubMed
        13. Li Q, Wright SE, Matijasevic Z, Chong W, Ludlum DB, Volkert MR. The role of human alkyladenine glycosylase in cellular resistance to the chloroethylnitrosoureas. Carcinogenesis. 2003 Mar; 24(3):589-93.
          View in: PubMed
        14. Bonanno K, Wyrzykowski J, Chong W, Matijasevic Z, Volkert MR. Alkylation resistance of E. coli cells expressing different isoforms of human alkyladenine DNA glycosylase (hAAG). DNA Repair (Amst). 2002 Jul 17; 1(7):507-16.
          View in: PubMed
        15. Volkert MR, Landini P. Transcriptional responses to DNA damage. Curr Opin Microbiol. 2001 Apr; 4(2):178-85.
          View in: PubMed
        16. Volkert MR, Elliott NA, Housman DE. Functional genomics reveals a family of eukaryotic oxidation protection genes. Proc Natl Acad Sci U S A. 2000 Dec 19; 97(26):14530-5.
          View in: PubMed
        17. Landini P, Volkert MR. Regulatory responses of the adaptive response to alkylation damage: a simple regulon with complex regulatory features. J Bacteriol. 2000 Dec; 182(23):6543-9.
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        18. Dunman PM, Ren L, Rahman MS, Palejwala VA, Murphy HS, Volkert MR, Humayun MZ. Escherichia coli cells defective for the recN gene display constitutive elevation of mutagenesis at 3,N(4)-ethenocytosine via an SOS-induced mechanism. Mol Microbiol. 2000 Aug; 37(3):680-6.
          View in: PubMed
        19. Landini P, Bown JA, Volkert MR, Busby SJ. Ada protein-RNA polymerase sigma subunit interaction and alpha subunit-promoter DNA interaction are necessary at different steps in transcription initiation at the Escherichia coli Ada and aidB promoters. J Biol Chem. 1998 May 22; 273(21):13307-12.
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        20. Adam E, Volkert MR, Blot M. Cytochrome c biogenesis is involved in the transposon Tn5-mediated bleomycin resistance and the associated fitness effect in Escherichia coli. Mol Microbiol. 1998 Apr; 28(1):15-24.
          View in: PubMed
        21. Landini P, Gaal T, Ross W, Volkert MR. The RNA polymerase alpha subunit carboxyl-terminal domain is required for both basal and activated transcription from the alkA promoter. J Biol Chem. 1997 Jun 20; 272(25):15914-9.
          View in: PubMed
        22. Landini P, Hajec LI, Nguyen LH, Burgess RR, Volkert MR. The leucine-responsive regulatory protein (Lrp) acts as a specific repressor for sigma s-dependent transcription of the Escherichia coli aidB gene. Mol Microbiol. 1996 Jun; 20(5):947-55.
          View in: PubMed
        23. Landini P, Volkert MR. RNA polymerase alpha subunit binding site in positively controlled promoters: a new model for RNA polymerase-promoter interaction and transcriptional activation in the Escherichia coli ada and aidB genes. EMBO J. 1995 Sep 1; 14(17):4329-35.
          View in: PubMed
        24. Landini P, Volkert MR. Transcriptional activation of the Escherichia coli adaptive response gene aidB is mediated by binding of methylated Ada protein. Evidence for a new consensus sequence for Ada-binding sites. J Biol Chem. 1995 Apr 7; 270(14):8285-9.
          View in: PubMed
        25. Volkert MR, Hajec LI, Matijasevic Z, Fang FC, Prince R. Induction of the Escherichia coli aidB gene under oxygen-limiting conditions requires a functional rpoS (katF) gene. J Bacteriol. 1994 Dec; 176(24):7638-45.
          View in: PubMed
        26. Landini P, Hajec LI, Volkert MR. Structure and transcriptional regulation of the Escherichia coli adaptive response gene aidB. J Bacteriol. 1994 Nov; 176(21):6583-9.
          View in: PubMed
        27. Volkert MR, Loewen PC, Switala J, Crowley D, Conley M. The delta (argF-lacZ)205(U169) deletion greatly enhances resistance to hydrogen peroxide in stationary-phase Escherichia coli. J Bacteriol. 1994 Mar; 176(5):1297-302.
          View in: PubMed
        28. Matijasevic Z, Hajec LI, Volkert MR. Anaerobic induction of the alkylation-inducible Escherichia coli aidB gene involves genes of the cysteine biosynthetic pathway. J Bacteriol. 1992 Mar; 174(6):2043-6.
          View in: PubMed
        29. Volkert MR, Hajec LI. Molecular analysis of the aidD6::Mu d1 (bla lac) fusion mutation of Escherichia coli K12. Mol Gen Genet. 1991 Oct; 229(2):319-23.
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        30. Volkert MR, Gately FH, Hajec LI. Expression of DNA damage-inducible genes of Escherichia coli upon treatment with methylating, ethylating and propylating agents. Mutat Res. 1989 Mar; 217(2):109-15.
          View in: PubMed
        31. Volkert MR, Hajec LI, Nguyen DC. Induction of the alkylation-inducible aidB gene of Escherichia coli by anaerobiosis. J Bacteriol. 1989 Feb; 171(2):1196-8.
          View in: PubMed
        32. Volkert MR. Altered induction of the adaptive response to alkylation damage in Escherichia coli recF mutants. J Bacteriol. 1989 Jan; 171(1):99-103.
          View in: PubMed
        33. Fram RJ, Crockett J, Volkert MR. Gene expression caused by alkylating agents and cis-diamminedichloroplatinum(II) in Escherichia coli. Cancer Res. 1988 Sep 1; 48(17):4823-6.
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        34. Poteete AR, Volkert MR. Activation of recF-dependent recombination in Escherichia coli by bacteriophage lambda- and P22-encoded functions. J Bacteriol. 1988 Sep; 170(9):4379-81.
          View in: PubMed
        35. Fram RJ, Marinus MG, Volkert MR. Gene expression in E. coli after treatment with streptozotocin. Mutat Res. 1988 Mar; 198(1):45-51.
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        36. Volkert MR. Adaptive response of Escherichia coli to alkylation damage. Environ Mol Mutagen. 1988; 11(2):241-55.
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        37. Volkert MR, Hartke MA. Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena. Mutat Res. 1987 Nov; 184(3):181-6.
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        38. Volkert MR, Nguyen DC, Beard KC. Escherichia coli gene induction by alkylation treatment. Genetics. 1986 Jan; 112(1):11-26.
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        39. Volkert MR, Margossian LJ, Clark AJ. Two-component suppression of recF143 by recA441 in Escherichia coli K-12. J Bacteriol. 1984 Nov; 160(2):702-5.
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        40. Volkert MR, Nguyen DC. Induction of specific Escherichia coli genes by sublethal treatments with alkylating agents. Proc Natl Acad Sci U S A. 1984 Jul; 81(13):4110-4.
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        41. Volkert MR, Hartke MA. Suppression of Escherichia coli recF mutations by recA-linked srfA mutations. J Bacteriol. 1984 Feb; 157(2):498-506.
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        42. Clark AJ, Volkert MR, Margossian LJ, Nagaishi H. Effects of a recA operator mutation on mutant phenotypes conferred by lexA and recF mutations. Mutat Res. 1982 Nov; 106(1):11-26.
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        43. Uhlin BE, Volkert MR, Clark AJ, Sancar A, Rupp WD. Nucleotide sequence of a recA operator mutation. LexA/operator-repressor binding/inducible repair. Mol Gen Genet. 1982; 185(2):251-4.
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        44. Witkin EM, McCall JO, Volkert MR, Wermundsen IE. Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli. Mol Gen Genet. 1982; 185(1):43-50.
          View in: PubMed
        45. Volkert MR, Margossian LJ, Clark AJ. Evidence that rnmB is the operator of the Escherichia coli recA gene. Proc Natl Acad Sci U S A. 1981 Mar; 78(3):1786-90.
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        46. Volkert MR, Spencer DF, Clark AJ. Indirect and intragenic suppression of the lexA102 mutation in E. coli B/r. Mol Gen Genet. 1979; 177(1):129-37.
          View in: PubMed
        47. Clark AJ, Volkert MR, Margossian LJ. A role for recF in repair of UV damage to DNA. Cold Spring Harb Symp Quant Biol. 1979; 43 Pt 2:887-92.
          View in: PubMed
        48. Volkert MR, George DL, Witkin EM. Partial suppression of the LexA phenotype by mutations (rnm) which restore ultraviolet resistance but not ultraviolet mutability to Escherichia coli B/r uvr A lexA. Mutat Res. 1976 Jul; 36(1):17-28.
          View in: PubMed
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