Isolation and analyses of MutY homologs (MYH).
The base excision repair carried out by the bacterial MutY DNA glycosylase and eukaryotic MutY homolog (MYH) is responsible for removing adenines misincorporated into DNA opposite 7,8-dihydro-8-oxo-guanines (8-oxoG), thereby preventing G:C to T:A mutations. MutY and MYH can also remove adenines from A/G and A/C and can remove guanines from G/8-oxoG mismatches at reduced rates. Biallelic germline mutations in the human MYH gene predispose individuals to multiple colorectal adenomas and carcinoma. Four functional assays are usually employed to characterize the MutY and MYH. Gel mobility shift or fluorescence anisotropy assays measures DNA-binding affinity and the apparent dissociation constants. Glycosylase assay determines the catalytic parameters of the enzyme. By using a trapping assay in the presence of sodium borohydride, the protein-DNA covalent intermediate can be identified. The in vivo activity of MutY or MYH can be measured by complementation in Escherichia coli mutY mutants or fission yeast Schizosaccharomyces pombe MYH knockout cells. MutY and MYH interacting proteins can be analyzed by the glutathione S-transferase pull-down assay, Far-western, and coimmunoprecipitation. The in vitro and in vivo activities of MYH can be modulated by several proteins, including mismatch recognition enzymes MSH2/MSH6, proliferating cell nuclear antigen, and apurinic/apyrimidinic endonuclease.
Lu-Chang
A-Lien
AL
University of Maryland, Department of Biochemistry, Baltimore, USA.
D000818:Animals; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004260:DNA Repair; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D005656:Fungal Proteins; D011994:Recombinant Proteins; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase