N-terminus of the rat adenine glycosylase MYH affects excision rates and processing of MYH-generated abasic sites.
Repair of most modified and mispaired bases in the genome is initiated by DNA glycosylases, which bind to their respective targets and cleave the N-glycosyl bond to initiate base excision repair (BER). The mammalian homolog of the Escherichia coli MutY DNA glycosylase (MYH) cleaves adenine residues paired with either oxidized or non-modified guanines. MYH is crucial for the avoidance of mutations resulting from oxidative DNA damage. Multiple N-terminal splice variants of MYH exist in mammalian cells and it is likely that different variants result in the production of enzymes with altered properties. To investigate whether modifications in the N-terminus are consequential to MYH function, we overexpressed intact and N-terminal-deletion rat MYH proteins and examined their activities. We found that deletion of 75 amino acids, which perturbs the catalytic core that is conserved with E.coli MutY, abolished excision activity. In contrast, deletions limited to the extended mammalian N-terminal domain, differentially influenced steady-state excision rates. Notably, deletion of 50 amino acids resulted in an enzyme with a significantly lower K(m) favoring formation of excision products with 3'-OH termini. Our findings suggest that MYH isoforms divergent in the N-terminus influence excision rates and processing of abasic sites.
Department of Surgery and Shriners Hospitals for Children, The University of Texas Medical Branch, 815 Market Street, Galveston, TX 77550, USA.
Huaxian
H
Ma
Heung M
HM
Lee
Ella W
EW
Englander
D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D017384:Sequence Deletion
D015636:Magnesium Chloride; D006147:Guanine; D004247:DNA; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D000225:Adenine