The proposed research deals with a very fundamental important question: """"""""Do the majority of the mutations induced in human cells arise as the result of special proteins carrying out error- prone translesion synthesis to bypass DNA damage that blocks fork progression? Until now, this question could not be answered. Mutations are considered to contribute significantly to the neoplastic transformation of human cells. If spontaneously arising or exogenous DNA damage is not repaired before S-phase replication, and the unrepaired damage interferes with fork progression, a process known as translesion bypass is considered to be invoked. In bacteria and lower eukaryotes, such translesion synthesis depends upon a set of proteins that differ, at least in part, from those used for normal chromosome replication. However, the nature of the process in mammalian cells is unknown. Very recently, C.W. Lawrence and his group at the Univ. of Rochester cloned the human homolog of the S. cerevisiae REV3 and Rev1 genes. Using a derivative of our infinite life span, near-diploid, karyotypically-stable cell strain, MSU-1.2, that expresses antisense hsREV3 under the control of a tetracycline promoter, we carried out a pilot study comparing the frequency of mutations induced by UV and in MSU-1.2 cells that do not express this antisense. The frequency of mutants induced in the cells with antisense hsREV was significantly lower. These results, although preliminary, raise the possibility that the majority of mutations induced in human cells by a variety of agents result from translesion synthesis by the human equivalent of scRev3p + scRev7p (polymerase zeta) and the scREV1 gene product. We will test this hypothesis in sets of human cell strains that express or do not express antisense hsREV3 and hsREV1 and cells in which the endogenous genes have or have not been eliminated by mutations. The agents to be tested include UV, BPDE, 1-NOP, N AcO-AAF, MNU and ENU, and Co60. These cell strains will be compared for the frequency and spectra of mutations induced in the HPRT gene. Cell-free extracts from these same strains, capable of replicating M13mp2 phage RF I containing damage induced by the first four agents will also be compared for the ability to by-pass the damage in the RF I DNA and for the frequency and spectra of mutations induced in the LacZ-alpha gene.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
1R01ES009822-01
Application #
2825998
Study Section
Special Emphasis Panel (ZRG1-MEP (03))
Program Officer
Okano, Paul
Project Start
1999-05-01
Project End
2003-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Michigan State University
Department
Microbiology/Immun/Virology
Type
Schools of Osteopathy
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Li, Ziqiang; Zhang, Hong; McManus, Terrence P et al. (2002) hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts. Mutat Res 510:71-80
Li, Ziqiang; Xiao, Wei; McCormick, J Justin et al. (2002) Identification of a protein essential for a major pathway used by human cells to avoid UV- induced DNA damage. Proc Natl Acad Sci U S A 99:4459-64
Lawrence, C W; Maher, V M (2001) Eukaryotic mutagenesis and translesion replication dependent on DNA polymerase zeta and Rev1 protein. Biochem Soc Trans 29:187-91
Lawrence, C W; Maher, V M (2001) Mutagenesis in eukaryotes dependent on DNA polymerase zeta and Rev1p. Philos Trans R Soc Lond B Biol Sci 356:41-6
Gibbs, P E; Wang, X D; Li, Z et al. (2000) The function of the human homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis induced by UV light. Proc Natl Acad Sci U S A 97:4186-91
Lawrence, C W; Gibbs, P E; Murante, R S et al. (2000) Roles of DNA polymerase zeta and Rev1 protein in eukaryotic mutagenesis and translesion replication. Cold Spring Harb Symp Quant Biol 65:61-9