期刊
出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2116218119
关键词
DNA repair; DNA alkyltransferase; single-molecule studies; fluorescence optical tweezers
资金
- Deutsche Forschungsgemeinschaft (DFG) [TE-671/4-2]
AGT is an important DNA repair protein that repairs highly mutagenic and cytotoxic alkylguanine lesions. Studies have revealed the interactions of AGT with alkyl lesions and its role in DNA repair mechanism. Surprisingly, AGT clusters do not enhance DNA translocation speed, suggesting a different role in AGT function.
The O6-alkylguanine DNA alkyltransferase (AGT) is an important DNA repair protein. AGT repairs highly mutagenic and cytotoxic alkylguanine lesions that result from metabolic products but are also deliberately introduced during chemotherapy, making a better understanding of the working mechanism of AGT essential. To investigate lesion interactions by AGT, we present a protocol to insert a single alkylguanine lesion at a well-defined position in long DNA substrates for single-molecule fluorescence microscopy coupled with dual-trap optical tweezers. Our studies address the longstanding enigma in the field of how monomeric AGT complexes at alkyl lesions seen in crystal structures can be reconciled with AGT clusters on DNA at high protein concentrations that have been observed from atomic force microscopy (AFM) and biochemical studies. A role of AGT clusters in enhancing lesion search efficiencies by AGT has previously been proposed. Surprisingly, our data show no enhancement of DNA translocation speed by AGT cluster formation, suggesting that AGT clusters may serve a different role in AGT function. Interestingly, a possible role of these clusters is indicated by preferential cluster formation at alkyl lesions in our studies. From our data, we derive a model for the lesion search and repair mechanism of AGT.
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