DISULPHIDE CROSS-LINKED RARE SEARCH INTERMEDIATE OF HOGG1 ON UNDAMAGED DNA

Cornell University, Ithaca NY

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The mechanism by which DNA glycosylases differentiate between specific damaged bases and the overwhelming excess of normal bases remains a mystery. Moreover, it is also unclear how these enzymes, despite spending so much time interrogating normal DNA, constrain themselves to cleaving only damaged bases. In particular, the feat of recognizing 8-oxoguanine lesions (oxoG) is impressive, because guanine and oxoG differ by merely two atoms, and the presence of oxoG in B-form DNA elicits no significant structural perturbation. Per our efforts to better understand these issues, we have used disulfide cross-linking technology (DXL) to covalently trap a human 8-oxoguanine glycosylase (hOGG1) to an undamaged G:C base pair in a normal duplex of DNA. To date, we have reported numerous structures of hOGG1/DNA complexes, several of which have borne DNA components having no lesion, but all have been configured so as to promote disruption of the target base-pair. Our success in obtaining the intrahelical G:C base pair in the case of MutM, a bacterial DNA glycosylase that recognizes the same lesion as hOGG1, came about systematically screening a number of suitable sites for introduction of a DXL, then crystallizing and solving structures of the resulting crosslinked complexes. We strongly believe that this same systematic screening strategy will prove successful in obtaining a structure of the hOGG1 DNA interrogation complex. With a new cross-linking site as well as a new disulfide tether that we have developed, we have already obtained promising hOGG1/DNA crystals.