Adenine removal activity and bacterial complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer

Abstract

MUTYH-associated polyposis (MAP) is the only inherited colorectal cancer syndrome that is associated with inherited biallelic mutations in a base excision repair gene. The MUTYH glycosylase plays an important role in preventing mutations associated with 8-oxoguanine (OG) by removing adenine residues that have been misincorporated opposite OG. MAP-associated mutations are present throughout MUTYH, with a large number coding for missense variations. To date the available information on the functional properties of MUTYH variants is conflicting. In this study, a kinetic analysis of the adenine glycosylase activity of MUTYH and several variants was undertaken using a correction for active fraction to control for differences due to overexpression and purification. Using these methods, the rate constants for steps involved in the adenine removal process were determined for the MAP variants Y165C, G382D, P391L and Q324R MUTYH. Under single-turnover conditions, the rate of adenine removal for these four variants was found to be 30-40% of WT MUTYH. In addition, the ability of MUTYH and the variants to suppress mutations and complement for the absence of MutY in E. coli was assessed using rifampicin resistance assays. The presence of WT and Q324R MUTYH resulted in complete suppression of the mutation frequency, while G382D MUTYH showed reduced ability to suppress the mutation frequency. In contrast, the mutation frequency observed upon expression of P391L and Y165C MUTYH were similar to the controls, suggesting no activity toward preventing DNA mutations. Notably, though all variations studied herein resulted in similar reductions in adenine glycosylase activity, the effects in the bacterial complementation are quite different. This suggests that the consequences of a specific amino acid variation on overall repair in a cellular context may be magnified.

A human MUTYH variant linking colonic polyposis to redox degradation of the [4Fe4S]2+ cluster

“…For a more complete functional comparison of the four MUTYH variants, we conducted time­course glycosylase assays under multiple turnover conditions in order to quantitatively determine the proportion of active enzyme in each sample[19, 20, 21]. These assays were performed using dsDNA containing an 8­oxoG:A mispair together with varying concentrations of MUTYH proteins.…”

Leiden open variation database of the MUTYH gene

“…3, 6, 7, 9, 10, 11, 12 A 3, 7, 11, 12 A/N 4, 5, 6, 13 IF, immunofluorescence; IHC, immunohistochemistry; RT-PCR, reverse transcriptase PCR; A, aberrant result; N, normal result and/or similar to wild type; 1, [Yamaguchi et al, 2002]; 2, [Chen et al, 2008]; 3, [Ali et al, 2008]; 4, [Di Gregorio et al, 2006]; 5, [O'Shea et al, 2008]; 6, [Molatore et al, 2010];7, [D'Agostino et al, 2010]; 8, [Fostira et al, 2010]; 9, [Chmiel et al, 2003]; 10, [Kundu et al, 2009]; 11, [Parker et al, 2005]; 12, [Wooden et al, 2004]; 13, [van der Post et al, 2009]; 14, [Dallosso et al, 2008]; 15, [Bai et al, 2005]; 16, [Bai et al, 2007]; 17, [Kanter-Smoler et al, 2006]; 18, [Tao et al, 2004]; 19, LOVD; 20, [Shinmura et al, 2000]; 21, …”

A Zinc Linchpin Motif in the MUTYH Glycosylase Interdomain Connector Is Required for Efficient Repair of DNA Damage

“…14 Low MUTYH-mediated repair allows for accumulation of mutations in an RNA polymerase, making rifampicin a less effective block to transcription. The mutation frequency ( f ) is related to the number of rifampicin resistant colonies.…”