Insight into the functional consequences of inherited variants of the hMYH adenine glycosylase associated with colorectal cancer: complementation assays with hMYH variants and pre-steady-state kinetics of the corresponding mutated E.coli enzymes.
The oxidized guanine lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is highly mutagenic, resulting in G:C to T:A transversion mutations in the absence of repair. The Escherichia coli adenine glycosylase MutY and its human homolog (hMYH) play an important role in the prevention of mutations associated with OG by removing misincorporated adenine residues from OG:A mismatches. Previously, biallelic mutations of hMYH have been identified in a British family (Family N) with symptoms characteristic of familial adenomatous polyposis (FAP), which is typically associated with mutations in the adenomatous polyposis coli (APC) gene. Afflicted members of this family were compound heterozygotes for two mutations in hMYH, Y165C and G382D. These positions are highly conserved in MutY across phylogeny. The current work reveals a reduced ability of the hMYH variants compared to wild-type (WT) hMYH to complement the activity of E.coli MutY in mutY((-)) E.coli. In vitro analysis of the corresponding mutations in E.coli MutY revealed a reduction in the adenine glycosylase activity of the enzymes. In addition, evaluation of substrate affinity using a substrate analog, 2'-deoxy-2'-fluoroadenosine (FA) revealed that both mutations severely diminish the ability to recognize FA, and discriminate between OG and G. Importantly, adenine removal with both the mutant and WT E.coli enzymes was observed to be less efficient from a mismatch in the sequence context observed to be predominantly mutated in tumors of Family N. Interestingly, the magnitude of the reduced activity of the E.coli mutant enzymes relative to the WT enzyme was magnified in the "hotspot" sequence context. If the corresponding mutations in hMYH cause similar sensitivity to sequence context, this effect may contribute to the specific targeting of the APC gene. The lack of complementation of the hMYH variants for MutY, and the reduced activity of the Y82C and G253D E.coli enzymes, provide additional circumstantial evidence that the somatic mutations in APC, and the occurrence of FAP in Family N, are due to a reduced ability of the Y165C and G382D hMYH enzymes to recognize and repair OG:A mismatches.
Chmiel
Nikolas H
NH
Department of Chemistry, University of Utah, Henry Erying Building, 315 S 1400 E RM, Salt Lake City 84112, USA.
Livingston
Alison L
AL
David
Sheila S
SS
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000225:Adenine; D011125:Adenomatous Polyposis Coli / Q000201:enzymology*; D020137:Base Pair Mismatch; D001665:Binding Sites; D015179:Colorectal Neoplasms / Q000201:enzymology*; D045647:DNA Glycosylases; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D003839:Deoxyadenosines / Q000494:pharmacology; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D017491:Genes, APC / Q000502:physiology; D005816:Genetic Complementation Test; D014644:Genetic Variation; D006147:Guanine; D006579:Heterozygote; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D003839:Deoxyadenosines; D006147:Guanine; C030964:2'-fluoro-2'-deoxyadenosine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D003849:Deoxyguanosine; D000225:Adenine