The 3 '-> 5 ' exonuclease of T4 DNA polymerase removes premutagenic alkyl mispairs and contributes to futile cycling at O-6-methylguanine lesions

We have studied the processing of O-6-methylguanine (m6G)-containing oligon ucleotides and N-methyl-N-nitrosourea (MNU)-treated DNA templates by the 3' --> 5' exonuclease of T4 DNA polymerase. In vitro biochemical analyses dem onstrate that the exonuclease can remove bases opposite a defined m6G lesio n. The efficiency of excision of a terminal m6G .T was similar to that of m 6G .C, and both mere excised as efficiently as a GT substrate. Partitioning assays between the polymerase and exonuclease activities, performed in the presence of dNTPs, resulted in repeated incorporation and excision events opposite the m6G lesion. This idling produces dramatically less full-length product, relative to natural substrates, indicating that the 3' --> 5' exo nuclease may contribute to DNA synthesis inhibition by alkylating agents. G enetic data obtained using an in vitro herpes simplex virus-thymidine kinas e assay support the inefficiency of the exonuclease as a "proofreading" act ivity for m6G, since virtually all mutations produced by the native enzyme using MNU-treated templates were G --> A transitions. Comparison of MNU dos e-response curves for exonuclease-proficient and -deficient forms of T4 pol ymerase reveals that the exonuclease efficiently removes 50-86% of total pr emutagenic alkyl mispairs. We propose that idling of exonuclease-proficient polymerases at m6G lesions during repair DNA synthesis provides the bioche mical explanation for cellular cytotoxicity of methylating agents.