Abstract
Biallelic inactivating germline mutations in the base excision repair MUTYH (MYH) gene have been shown to predispose to MUTYH-associated polyposis (MAP), which is characterized by multiple colorectal adenomas and carcinomas. In this study, we successfully prepared highly homogeneous human MUTYH type 2 recombinant proteins and compared the DNA glycosylase activity of the wild-type protein and fourteen variant-type proteins on adenine mispaired with 8-hydroxyguanine, an oxidized form of guanine. The adenine DNA glycosylase activity of the p.I195V protein, p.G368D protein, p.M255V protein, and p.Y151C protein was 66.9%, 15.2%, 10.7%, and 4.5%, respectively, of that of the wild-type protein, and the glycosylase activity of the p.R154H, p.L360P, p.P377L, p.452delE, p.R69X, and p.Q310X proteins as well as of the p.D208N negative control form was extremely severely impaired. The glycosylase activity of the p.V47E, p.R281C, p.A345V, and p.S487F proteins, on the other hand, was almost the same as that of the wild-type protein. These results should be of great value in accurately diagnosing MAP and in fully understanding the mechanism by which MUTYH repairs DNA in which adenine is mispaired with 8-hydroxyguanine. © 2010 Wiley-Liss, Inc.
Highlights
8-Hydroxyguanine (8-OHG) is an oxidized form of guanine (Kasai and Nishimura, 1991), and because 8-OHG can pair with adenine as well as cytosine, formation of 8-OHG in DNA causes a G:C to T:A transversion mutation (Shibutani et al, 1991)
Their molecular size of approximately 61 kDa was determined by SDS-polyacrylamide gel electrophoresis (PAGE) / Coomassie Brilliant Blue (CBB) staining and Western blotting, and it corresponded to their size calculated from the cDNA sequence
The results showed that highly purified wild-type MUTYH type 2 protein had been obtained with our expression and purification system, and the adenine DNA glycosylase activity of the protein on an A:8-OHG mispair was satisfactorily detected by our assay
Summary
8-Hydroxyguanine (8-OHG) is an oxidized form of guanine (Kasai and Nishimura, 1991), and because 8-OHG can pair with adenine as well as cytosine, formation of 8-OHG in DNA causes a G:C to T:A transversion mutation (Shibutani et al, 1991). Even though more than 80 MUTYH variants have been described in the MUTYH gene in colorectal polyposis and colorectal cancer patients (reviewed in Cheadle and Sampson, 2007; Vogt et al, 2009), the effect of only a small number of variations on human MUTYH protein activity has been investigated (Wooden et al, 2004; Bai et al, 2005; Bai et al, 2007; Ali et al, 2008; Kundu et al, 2009; Forsbring et al, 2009; Molatore et al, 2010). Since somatic APC (MIM# 611731) mutations occur in the nuclear DNA of a high proportion of MAP tumors (Al-Tassan et al, 2002), it is preferable to evaluate the repair activity of the type 2 protein localized in the nucleus, not the type 1 mitochondrial protein. This study assessed the adenine excisional activity of a larger number of MUTYH variants than in previous studies, and the repair activity of the type 2 protein of 11 of the 14 MUTYH variants (p.V47E, p.R154H, p.I195V, p.M255V, p.R281C, p.A345V, p.L360P, p.P377L, p.S487F, p.R69X, and p.Q310X) was examined for the first in this study
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