A distinct TthMutY bifunctional glycosylase that hydrolyzes not only adenine but also thymine opposite 8-oxoguanine in the hyperthermophilic bacterium, Thermus thermophilus.
Oxidative damage represents a major threat to genomic stability because the major product of DNA oxidation, 8-oxoguanine (GO), frequently mispairs with adenine during replication. We were interested in finding out how hyperthermophilic bacteria under goes the process of excising mispaired adenine from A/GO to deal with genomic oxidative damage. Herein we report the properties of an Escherichia coli MutY (EcMutY) homolog, TthMutY, derived from a hyperthermophile Thermus thermophilus. TthMutY preferentially excises on A/GO and G/GO mispairs and has additional activities on T/GO and A/G mismatches. TthMutY has significant sequence homology to the A/G and T/G mismatch recognition motifs, respectively, of MutY and Mig.MthI. A substitution from Tyr112 to Ser or Ala (Y112S and Y112A) in the putative thymine-binding site of TthMutY showed significant decrease in DNA glycosylase activity. A mutant form of TthMutY, R134K, could form a Schiff base with DNA and fully retained its DNA glycosylase activity against A/GO and A/G mispair. Interestingly, although TthMutY cannot form a trapped complex with substrate in the presence of NaBH(4), it expressed AP lyase activity, suggesting Tyr112 in TthMutY may be the key residue for AP lyase activity. These results suggest that TthMutY may be an example of a novel class of bifunctional A/GO mismatch DNA glycosylase that can also remove thymine from T/GO mispair.
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Jung Ho
JH
Department of Advanced Technology Fusion, BMIC, Konkuk University, Gwangjin-gu, Seoul 143-701, Republic of Korea.
Park
Jong Hwa
JH
Chung
Ji Hyung
JH
Kim
Darrick S H L
DS
Han
Ye Sun
YS
D000225:Adenine / Q000378:metabolism*; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045647:DNA Glycosylases / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics; D013941:Thymine / Q000378:metabolism*
D017931:DNA Primers; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D000225:Adenine; D013941:Thymine