Hypersensitivity of DNA polymerase beta null mouse fibroblasts reflects accumulation of cytotoxic repair intermediates from site-specific alkyl DNA lesions.
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.
Horton
Julie K
JK
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Joyce-Gray
Donna F
DF
Pachkowski
Brian F
BF
Swenberg
James A
JA
Wilson
Samuel H
SH
D000477:Alkylating Agents / Q000378:metabolism; D000478:Alkylation; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000502:physiology; D004249:DNA Damage / Q000235:genetics* / Q000502:physiology; D019951:DNA Polymerase beta / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics* / Q000502:physiology; D005347:Fibroblasts; D066298:In Vitro Techniques; D051379:Mice; D009429:Netropsin / Q000031:analogs & derivatives* / Q000378:metabolism; D013997:Time Factors
D000477:Alkylating Agents; D000970:Antineoplastic Agents; C110963:methyl lexitropsin; D009429:Netropsin; D004247:DNA; D019951:DNA Polymerase beta