Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity, glycosylase activity and interaction with PCNA and Hus1

Abstract

MUTYH is a base excision repair (BER) protein that prevents mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenine from inappropriately formed OG:A base-pairs. Germline mutations in the MUTYH gene are linked to colorectal polyposis and a high risk of colorectal cancer, a syndrome referred to as MUTYH-associated polyposis (MAP). There are over 300 different MUTYH mutations associated with MAP and a large fraction of these gene changes code for missense MUTYH variants. Herein, the adenine glycosylase activity, mismatch recognition properties, and interaction with relevant protein partners of human MUTYH and five MAP variants (R295C, P281L, Q324H, P502L, and R520Q) were examined. P281L MUTYH was found to be severely compromised both in DNA binding and base excision activity, consistent with the location of this variation in the iron-sulfur cluster (FCL) DNA binding motif of MUTYH. Both R295C and R520Q MUTYH were found to have low fractions of active enzyme, compromised affinity for damaged DNA, and reduced rates for adenine excision. In contrast, both Q324H and P502L MUTYH function relatively similarly to WT MUTYH in both binding and glycosylase assays. However, P502L and R520Q exhibited reduced affinity for PCNA (proliferation cell nuclear antigen), consistent with their location in the PCNA-binding motif of MUTYH. Whereas, only Q324H, and not R295C, was found to have reduced affinity for Hus1 of the Rad9-Hus1-Rad1 complex, despite both being localized to the same region implicated for interaction with Hus1. These results underscore the diversity of functional consequences due to MUTYH variants that may impact the progression of MAP.

Repair of 8-oxoG:A mismatches by the MUTYH glycosylase: Mechanism, metals and medicine

“…These experiments illustrate that the activity of G382D MUTYH is more dramatically reduced within cells than the in vitro experiments indicated. This may be due in part to the more difficult task of repair in a cellular context [82, 87, 134], as well as other features that may come into play in a cellular context, such as interactions and competition with other proteins [88, 111]. These studies underscore the importance of multiple assays, along with structural studies, to fully explore the functional implications of specific variants.…”

“…Using a similar approach, Mazzei and co-workers have found that expression of Q324H in cells was associated with 2-fold higher levels of 8-oxoG in DNA, hypersensitivity to oxidants and accumulation of the population in the S phase of the cell cycle [143]. Q324H is found in the IDC of MUTYH, an area of the protein not directly involved in DNA binding or catalysis, but known to facilitate key repair protein interactions with the Rad9-Hus1-Rad1 (9-1-1) complex and APE1 [134]. Q324H was shown to have reduced affinity for Hus-1 of the 9-1-1 complex compared to the WT protein [134, 144].…”

“…Q324H is found in the IDC of MUTYH, an area of the protein not directly involved in DNA binding or catalysis, but known to facilitate key repair protein interactions with the Rad9-Hus1-Rad1 (9-1-1) complex and APE1 [134]. Q324H was shown to have reduced affinity for Hus-1 of the 9-1-1 complex compared to the WT protein [134, 144]. Importantly, altered interactions with protein partners such as the 9-1-1 complex may result in MAP due to improper signaling responses to DNA damage [23, 143].…”

“…Functional studies in vitro have shown that P502L has similar DNA binding and glycosylase activity to WT enzyme, while binding affinity interactions with PCNA are dramatically reduced, which may more dramatically affect cellular repair. R520Q on the other hand was shown to have significantly reduced substrate binding, glycosylase activity and PCNA binding relative to WT [134]. …”

“…The second cofactor is a recently identified Zn 2+ ion, coordinated to three Cys residues in the interdomain connector (IDC) of higher eukaryotic MutY homologs; this region of MUTYH has been dubbed the “Zn 2+ linchpin motif” to embody its structural and functional roles [14]. These two MUTYH cofactors are essential for efficient repair in cells, but their exact role in DNA repair and regulation is still under investigation [14, 134, 150, 151]. …”