Abstract
The highly mutagenic A:8-oxoguanine (oxoG) base pair is generated mainly by misreplication of the C:oxoG base pair, the oxidation product of the C:G base pair. The A:oxoG base pair is particularly insidious because neither base in it carries faithful information to direct the repair of the other. The bacterial MutY (MUTYH in humans) adenine DNA glycosylase is able to initiate the repair of A:oxoG by selectively cleaving the A base from the A:oxoG base pair. The difference between faithful repair and wreaking mutagenic havoc on the genome lies in the accurate discrimination between two structurally similar base pairs: A:oxoG and A:T. Here we present two crystal structures of the MutY N-terminal domain in complex with either undamaged DNA or DNA containing an intrahelical lesion. These structures have captured for the first time a DNA glycosylase scanning the genome for a damaged base in the very first stage of lesion recognition and the base extrusion pathway. The mode of interaction observed here has suggested a common lesion-scanning mechanism across the entire helix-hairpin-helix superfamily to which MutY belongs. In addition, small angle X-ray scattering studies together with accompanying biochemical assays have suggested a possible role played by the C-terminal oxoG-recognition domain of MutY in lesion scanning.
Highlights
The highly mutagenic A:8-oxoguanine base pair is generated mainly by misreplication of the C:oxoG base pair, the oxidation product of the C:G base pair
We were able to place the N-terminal domain (NTD) in the electron density maps obtained from the crystals using molecular replacement, the position of the C-terminal domain (CTD) could not be assigned because of its poor electron density, which suggested that the CTD either exists in multiple conformations or is completely disordered in the Lesion Scanning Complex (LSC)
After screening several DNA sequences and using the cross-linking site P164C, which is well validated by the Lesion Recognition Complex (LRC) and anti-substrate complex (ASC) structures, we obtained a stable MutY NTD-DNA complex by cross-linking the lesion recognitioncompetent but catalytically inactive D144N mutant version of the MutY NTD to a 10-mer undamaged DNA containing a thiol attached to the A3 nucleobase in the major groove (Fig. 1, D and E)
Summary
Full-length MutY [14, 15, 18]. The C-terminal domain (CTD) of MutY structurally resembles MutT, an 8-oxo-dGTPase, and in MutY is responsible for recognition of the oxoG nucleobase, providing the means by which MutY discriminates oxoG from T [16, 17, 19]. In a related LRC structure having a wild-type active site but uncleavable synthetic nucleobase, 2Ј-fluoro-2Ј-deoxyadenosine (fluorinated lesion-recognition complex (FLRC)), the substrate base is plunged more deeply into the active pocket than in the LRC and forms more extensive interactions with active site residues, suggesting that adenine excision proceeds via general acid catalysis [13, 18, 22]. We have determined the structure of the MutY anti-substrate complex (ASC), in which the wild-type MutY is bound to DNA containing an oxoG:C base pair [19, 20]. We determined the crystal structures of the N-terminal domain of MutY bound to either undamaged DNA or DNA bearing an intrahelical oxoG:A base pair, which are named the N-terminal lesion scanning complex (N-LSC) and the N-terminal intrahelical lesion recognition complex (NILRC), respectively. The N-ILRC structure suggests a possible mechanism for how MutY first detects an oxoG:A base pair while it is fully intrahelical
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