Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

doi:10.1073/pnas.092662499

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

Laboratories of ^*Structural Biology and ^†Environmental Carcinogenesis and Mutagenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; ^‡Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599; ^§Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, TX 77555; and ^¶Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved March 13, 2002 (received for review December 11, 2001)

Abstract

The long-term effect of exposure to DNA alkylating agents is entwined with the cell's genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this “gene–environment interaction” paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA β-polymerase (β-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene–environment interaction. The β-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in β-pol null cells, but not in isogenic wild-type cells; UV light exposure or N-methyl-N′-nitro-N-nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in β-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in β-pol null cells eliminates the effect. In contrast, β-pol/AAG double null cells are slightly more mutable than the β-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure.

Footnotes

↵ ‖ To whom reprint requests should be addressed. E-mail: wilson5{at}niehs.nih.gov.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

AP,

apurinic/apyrimidinic;

BER,

base excision repair;

MMS,

methyl methanesulfonate;

dRP,

5′ deoxyribosephosphate;

β-pol,

DNA β-polymerase;

QPCR,

quantitative PCR;

AAG,

alkyladenine DNA glycosylase;

MEF,

mouse embryonic fibroblast;

MNNG,

N-methyl-N′-nitro-N-nitrosoguanidine;

pfu,

plaque-forming units