DNA SEARCH AND BASE FLIPPING MECHANISMS OF DNA GLYCOSYLASE, MUTM

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. MutM is a bacterial DNA glycosylase that specifically recognizes oxidatively damaged DNA bases and initiates the base excision repair pathway. Given the subtle conformational differences in the lesion base, 8-oxoguanine, and normal DNA base, guanine, and the scarcity of the lesions, a fundamental question arises: how does MutM locate the rare lesion sites among the vast genome? In addition, base flipping, an essential step for DNA glycosylase catalysis, is not well understood, and whether protein is actively promoting base extrusion is currently in debate. Using the disulfide crosslinking strategy, we have covalently trapped ordinarily transient intermediates during the search by MutM for lesions during nucleobase extrusion. These intermediates not only shed light on the mechanism of lesion recognition and catalysis, but have also provided valuable starting points for our computational studies of the base extrusion pathway.