Nunoshiba Tatsuo, Ishida Rikiya, Sasaki Michi, Iwai Shigenori, Nakabeppu Yusaku, Yamamoto Kazuo
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan.
Nucleic Acids Res. 2004 Oct 8;32(18):5339-48. doi: 10.1093/nar/gkh868. Print 2004.
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.
利用Nudix框在NCBI-BLAST数据库中搜索酿酒酵母中大肠杆菌MutT功能同源物的候选基因。Nudix框是一段在大肠杆菌MutT、普通假单胞菌MutT以及人、大鼠和小鼠的MTH1中保守的短氨基酸序列。在五个候选基因中，我们重点关注开放阅读框YLR151c，因为它有一个区域与MutT的N端一半包括Nudix框具有约76%的相似性。因此，我们评估了YLR151c作为大肠杆菌MutT在酿酒酵母中的功能同源物的能力。YLR151c的表达能够抑制大肠杆菌mutT缺陷菌株中由于氧化核苷酸8-氧代-dGTP错掺入导致的从A:T到C:G的颠换。与野生型相比，酵母菌株中YLR151c的缺失导致自发突变频率增加约14倍。此外，生化分析表明，GST-YLR151c融合蛋白对7,8-二氢-8-氧代-2'-脱氧鸟苷三磷酸（8-氧代-dGTP）和1,2-二氢-2-羟基-2'-脱氧腺苷三磷酸（2-OH-dATP）均具有焦磷酸酶活性。GST-YLR151c对8-氧代-dGTP的比活性为5.6×10⁻³ μM⁻¹ s⁻¹，这与大肠杆菌中MutT的备用酶RibA相似，但比hMTH1低150倍。从这些结果中，我们得出结论，YLR151c具有通过清除氧化核苷酸来预防自发诱变的能力，并且它可能是酿酒酵母中大肠杆菌MutT的功能同源物。
