Hypersensitivity of DNA polymerase beta null mouse fibroblasts reflects accumulation of cytotoxic repair intermediates from site-specific alkyl DNA lesions

DNA Repair (Amst). 2003 Jan 2;2(1):27-48. doi: 10.1016/s1568-7864(02)00184-2.

Abstract

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.

MeSH terms

  • Alkylating Agents / metabolism
  • Alkylation
  • Animals
  • Antineoplastic Agents / metabolism
  • Cell Line
  • DNA / physiology
  • DNA Damage / genetics*
  • DNA Damage / physiology
  • DNA Polymerase beta / genetics*
  • DNA Polymerase beta / metabolism
  • DNA Repair / genetics*
  • DNA Repair / physiology
  • Fibroblasts
  • In Vitro Techniques
  • Mice
  • Netropsin / analogs & derivatives*
  • Netropsin / metabolism
  • Time Factors

Substances

  • Alkylating Agents
  • Antineoplastic Agents
  • methyl lexitropsin
  • Netropsin
  • DNA
  • DNA Polymerase beta