Mutagens act through two distinct pathways: the first is through the direct misreplication of DNA damage inflicted by the mutagen. The second is an indirect pathway in which the mutagens alter the cellular physiology such as to enhance mutagenesis not only at the cognate DNA lesions, but also at other (heterologous) DNA damage present in the genome, and at undamaged cells. The Escherichia coli SOS phenomenon represents the best known inducible mutagenic pathway. We have recently described UVM, an SOS-independent DNA damage-inducible mutagenic response in Escherichia coli. The hypothesis in this proposal is that DNA damage in E. coli induces two parallel phenomena, namely, SOS and UvM, that play non-overlapping roles in inducible mutagenesis. UVM is proposed to modulate base insertion at noncoding lesions (UV modulation of mutagenesis), whereas the SOS response acts to bypass sites of stalled replication. Class 1 DNA lesions such as abasic sites require SOS functions for the bypass step, whereas Class 2 lesions (3,N4- ethenocytosine, epsilonC) do not. Two predictions of this hypothesis will be tested using a high resolution experimental system in which M13 viral single-stranded DNA molecules bearing defined site-specific DNA lesions will be used to probe the mutation fixation environment of the cell. Mutagenesis at the lesion in vivo and in vitro will be analyzed by a sequencing technology that does not require phenotype-based selection or screening for mutants. To test the prediction that the expression of SOS proteins at induced levels will be necessary but not sufficient to account for inducible error-prone replication observed in UV-irradiated cells, E. coli strains in which appropriate alleles of the recA, umuD and umuC genes are placed under the control of regulatable heterologous promoters will be constructed. Mutation fixation will be characterized at Class 1 and Class 2 lesions uninduced and UVM-induced cells in which the expression of SOS proteins is uncoupled from DNA damage. Whether DNA replication is altered in UVM induced cells will be tested by seeking the presence of error-prone replication capability in cell-free extracts prepared from UVM-induced cells. UVM-constitutive E. coli mutants recently isolated will be characterized as a first step toward isolating UVM-defective cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA073026-01
Application #
2011067
Study Section
Metabolic Pathology Study Section (MEP)
Project Start
1997-05-01
Project End
2001-02-28
Budget Start
1997-05-01
Budget End
1998-02-28
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
605799469
City
Newark
State
NJ
Country
United States
Zip Code
07107
Al Mamun, Abu Amar M (2007) Elevated expression of DNA polymerase II increases spontaneous mutagenesis in Escherichia coli. Mutat Res 625:29-39
Wang, Ge; Alamuri, Praveen; Humayun, M Zafri et al. (2005) The Helicobacter pylori MutS protein confers protection from oxidative DNA damage. Mol Microbiol 58:166-76
Dunman, P M; Ren, L; Rahman, M S et al. (2000) 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 37:680-6
Ren, L; Mamun, A A; Humayun, M Z (2000) Requirement for homologous recombination functions for expression of the mutA mistranslator tRNA-induced mutator phenotype in Escherichia coli. J Bacteriol 182:1427-31
Al Mamun, A A; Yadava, R S; Ren, L et al. (2000) The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro. Mol Microbiol 38:368-80
Rahman, M S; Humayun, M Z (1999) SOS and UVM pathways have lesion-specific additive and competing effects on mutation fixation at replication-blocking DNA lesions. J Bacteriol 181:1515-23
Al Mamun, A A; Rahman, M S; Humayun, M Z (1999) Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA-dependent error-prone DNA replication activity. Mol Microbiol 33:732-40
Ren, L; Al Mamun, A A; Humayun, M Z (1999) The mutA mistranslator tRNA-induced mutator phenotype requires recA and recB genes, but not the derepression of lexA-regulated functions. Mol Microbiol 32:607-15
Wang, G; Rahman, M S; Humayun, M Z (1997) Replication of M13 single-stranded viral DNA bearing single site-specific adducts by escherichia coli cell extracts: differential efficiency of translesion DNA synthesis for SOS-dependent and SOS-independent lesions. Biochemistry 36:9486-92
Murphy, H S; Humayun, M Z (1997) Escherichia coli cells expressing a mutant glyV (glycine tRNA) gene have a UVM-constitutive phenotype: implications for mechanisms underlying the mutA or mutC mutator effect. J Bacteriol 179:7507-14