Structural Studies of the Bacterial MutY Adenine DNA Glycosylase and Its Human Homolog MUTYH

Abstract

Cellular DNA is under constant threat of reactive oxygen species (ROS) produced by either endogenous or exogenous sources. One of the major forms of DNA damages caused by ROS is 8-oxoguanine (oxoG), which is highly mutagenic, although only differing from its progenitor guanine by two atoms. Oxidation of guanine generates the primary lesion oxoG:C, which upon misreplication produces oxoG:A. The oxoG:A base-pair is particularly insidious, because neither of the constituent nucleobases faithfully transmit genetic information from the original G:C base-pair. Repair of oxoG:A damage is initiated by adenine DNA glycosylases, which catalyzes hydrolytic cleavage of the aberrant adenine from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, are able to recognize the oxoG:A base-pair among a vast majority of normal base-pairs, and scrupulously avoid processing either the oxoG:C base-pair, or the T:A base-pair, because cleavage of the C residue would promote the mutagenic conversion to oxoG:A, and cleaving the A residue in the T:A base-pair would wreak havoc to the genome.

The previously published crystal structure of the MutY lesion recognition complex (LRC) has shed light on the molecular mechanism of damage recognition in the late stage of base-extrusion, where the target A is extruded into the active-site pocket of the enzyme. However, it is less well understood how MutY interacts with DNA (undamaged or damaged) in earlier stages of base-extrusion. In addition, the mechanism by which MutY precisely avoids cleaving C from oxoG:C has also remained a mystery. In this thesis, we report the crystal structures of several MutY-DNA complexes. Specifically, the N-terminal lesion scanning complex (N-LSC) and the N-terminal intrahelical lesion recognition complex (N-ILRC) capture the N-terminal domain (NTD) of MutY interacting with either undamaged DNA or DNA containing an intrahelical oxoG:A base-pair in the earliest stage of base-extrusion. Small-angle X-ray scattering (SAXS) analysis of the full-length MutY LSC has suggested the position of the C-terminal domain (CTD) in the LSC and the role of CTD in lesion scanning. The anti-substrate complex (ASC) and the ASC with a inactivating mutation (D144N ASC) each illustrates one of the two mechanisms by which MutY avoids cleaving the C residue in oxoG:C.

Besides our work on the bacterial MutY, we have also overexpressed and purified soluble MUTYH that contains all three core domains for the first time. In vitro adenine glycosylase assay has proven the purified protein active, and SAXS experiment has provided the first solution structure of MUTYH.