A novel Nudix hydrolase for oxidized purine nucleoside triphosphates encoded by ORFYLR151c (PCD1 gene) in Saccharomyces cerevisiae.
A search for candidates for a functional homologue of Escherichia coli MutT in yeast Saccharomyces cerevisiae was made in the NCBI-BLAST database using the Nudix box, a short amino acid sequence conserved among E.coli MutT, Pseudomonoas vulgaris MutT, and human, rat and mouse MTH1. Among five candidates, we focused on the open reading frame YLR151c, because it had a region with approximately 76% similarity to the N-terminal half of MutT including the Nudix box. We thus evaluated the ability of YLR151c as a functional homologue of E.coli MutT in S.cerevisiae. Expression of YLR151c was able to suppress the transversion from A:T to C:G caused by misincorporation of the oxidized nucleotide 8-oxo-dGTP in the E.coli mutT-deficient strain. The disruption of the YLR151c in yeast strain caused approximately 14-fold increase in the frequency of spontaneous mutation compared to the wild type. Additionally, biochemical analysis indicated that GST-YLR151c fusion protein possessed pyrophosphatase activity for both 7,8-dihydro-8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP) and 1,2-dihydro-2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP). The specific activity of GST-YLR151c for 8-oxo-dGTP was 5.6 x 10(-3) microM(-1) s(-1), which was similar to that of RibA, a backup enzyme for MutT in E.coli, but was 150-fold lower than that of hMTH1. From these results, we conclude that YLR151c has an ability to prevent spontaneous mutagenesis via sanitization of oxidized nucleotides, and that it may be the functional homologue of E.coli MutT in S.cerevisiae.
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan. tnuno@mail.tains.tohoku.ac.jp
Tatsuo
T
Nunoshiba
Rikiya
R
Ishida
Michi
M
Sasaki
Shigenori
S
Iwai
Yusaku
Y
Nakabeppu
Kazuo
K
Yamamoto
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D011685:Purine Nucleotides / Q000378:metabolism*; D011755:Pyrophosphatases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000494:pharmacology; D013489:Suppression, Genetic; D000096964:Nudix Hydrolases
D000255:Adenosine Triphosphate; D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; D010744:Phosphoric Monoester Hydrolases; D011685:Purine Nucleotides; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D012965:Sodium Chloride; D000096964:Nudix Hydrolases; C095141:2-hydroxydeoxyadenosine triphosphate; C078206:8-oxodeoxyguanosine triphosphate; C052211:mutT protein, E coli