Direct Observation of MUTYH and the Cancer-Associated Y150C Variant Binding to OG: A Mismatches at the Single Molecule Level

Abstract

Of the four DNA bases, guanine is the most likely to be damaged by reactive oxygen species. Oxidized guanine (specifically, 8-oxoguanine) mispairs with adenine (instead of cytosine) during DNA replication (an OG:A mismatch), ultimately resulting in genomic G -> T transversion mutations. MUTYH is a mammalian DNA repair glycosylase that locates and removes the adenine in these OG:A mismatches. Y150C is the mouse homolog of a human MUTYH variant that is associated with colorectal adenomas and carcinomas. Structural information indicates that tyrosine 150 is analogous to the critical “wedge residue” present in many other DNA glycosylases, which is essential for lesion recognition. Here, we directly observe, at the single molecule level in vitro, fluorescently tagged wildtype (WT) MUTYH and the Y150C variant while they scan suspended DNA “tightropes” comprised of concatenated, linearized plasmids that contain specifically positioned OG:A mismatches every 2700 base pairs. In bulk assays, Y150C demonstrates reduced catalytic activity. Although we find that both the WT and Y150C diffuse along undamaged DNA with the same diffusion constant (1x105nm2/s) as they scan for damage, Y150C remains bound to and scans the undamaged DNA for a much shorter period of time than WT (3.9s and 32.7s, respectively). Both the WT and Y150C recognize and pause at OG:A mismatches, suggesting that Y150C lesion recognition is not impaired. However, once recognized Y150C pauses at a mismatch for only a short time (median 1.9s), while the WT enzyme remains bound at these mismatches for at least the duration of our 5-minute recordings. Taken together, our data show that mutation of the tyrosine “wedge residue” does not impair MUTYH's ability recognize and pause at OG:A mismatches, although the variant is unable to form a long-lived complex.