Monofunctional
alkylating agents react with
DNA by S(N)1 or S(N)2 mechanisms resulting in formation of a wide spectrum of cytotoxic base adducts.
DNA polymerase beta (beta-pol) is required for efficient base excision repair of N-alkyl adducts, and we make use of the
hypersensitivity of beta-pol null mouse fibroblasts to investigate such
alkylating agents with a view towards understanding the DNA lesions responsible for the cellular phenotype. The inability of
O(6)-benzylguanine to sensitize wild-type or beta-pol null cells to S(N)1-type methylating agents indicates that the observed
hypersensitivity is not due to differential repair of cytotoxic O-alkyl adducts. Using a 3-methyladenine-specific agent and an inhibitor of such methylation, we find that inefficient repair of
3-methyladenine is not the reason for the
hypersensitivity of beta-pol null cells to methylating agents, and further that
3-methyladenine is not the adduct primarily responsible for
methyl methanesulfonate (MMS)- and methyl nitrosourea-induced cytotoxicity in wild-type cells. Relating the expected spectrum of
DNA adducts and the relative sensitivity of cells to monofunctional
alkylating agents, we propose that the
hypersensitivity of beta-pol null cells reflects accumulation of cytotoxic repair intermediates, such as the 5'-deoxyribose
phosphate group, following removal of 7-alkylguanine from
DNA. In support of this conclusion, beta-pol null cells are also hypersensitive to the
thymidine analog
5-hydroxymethyl-2'-deoxyuridine (hmdUrd). This agent is incorporated into cellular
DNA and elicits cytotoxicity only when removed by glycosylase-initiated base excision repair. Consistent with the hypothesis that there is a common repair intermediate resulting in cytotoxicity following treatment with both types of agents, both MMS and hmdUrd-initiated cell death are preceded by a similar rapid concentration-dependent suppression of
DNA synthesis and a later cell cycle arrest in G(0)/G(1) and G(2)M phases.