A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides

MUT相关蛋白在核苷酸池中降解DNA合成的诱变底物8-氧-7，8-二氢脱氧鸟苷三磷酸(8-oxo-dGTP)，从而防止DNA复制错误。在GenBank EST数据库的检索中，我们发现了一个新的MUT相关蛋白成员MTH2，它具有23个氨基酸的MUT模块。将克隆的小鼠MTH2(MMTH2)基因在大肠杆菌MUT(-)细胞中表达，并对其蛋白进行纯化。MMTH2蛋白可将8-oxo-dGTP水解为8-oxo-dGMP，其Km值为32&mgr；M。mMTH2基因的表达显著降低了大肠杆菌MUT(-)细胞自发突变频率的升高水平。因此，MTH2有可能保护遗传物质免受内源性氧自由基的不良影响。MTH2可以作为MTH1的冗余因子。此外，我们还报道了人类细胞有另一种清理核苷酸池的机制，以确保准确的DNA复制。人类Nudex Type 5(NUDT5)蛋白将8-oxo-dGDP水解为单磷酸，K(M)为0.77微米，大大低于ADP糖的K(M)，后者最初被确定为NUDT5的底物。NUDT5只能在很低的水平上水解8-oxo-dGTP，但当MUT有缺陷时，它可以替代MUT。当NUDT5在大肠杆菌MUT(-)细胞中表达时，自发突变的增加频率降低到正常水平。考虑到氧化鸟嘌呤核苷酸的MTH1和NUDT5的酶参数，NUDT5可能比MTH1在防止人类细胞中8-氧鸟嘌呤错误结合所导致的突变发生方面发挥更大的作用。

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool, thereby preventing DNA replication errors. During a search of GenBank EST database, we found a new member of MutT-related protein, MTH2, which possesses the 23-amino acid MutT module. The cloned mouse MTH2 (mMTH2) cDNA was expressed in Escherichia coli mutT(-) cells and the protein was purified. mMTH2 protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32&mgr;M. Expression of cDNA for mMTH2 reduced significantly the elevated level of spontaneous mutation frequency of E. coli mutT(-) cells. Thus, MTH2 has a potential to protect the genetic material from the untoward effects of endogenous oxygen radicals. MTH2 could act as an MTH1 redundancy factor.Also, we reported that human cells have another mechanism for cleaning up the nucleotide pool to ensure accurate DNA replication. The human Nudix type 5 (NUDT5) protein hydrolyses 8-oxo-dGDP to monophosphate with a K(m) of 0.77 microM, a value considerably lower than that for ADP sugars, which were originally identified as being substrates of NUDT5. NUDT5 hydrolyses 8-oxo-dGTP only at very low levels, but is able to substitute for MutT when it is defective. When NUDT5 is expressed in E. coli mutT(-) cells, the increased frequency of spontaneous mutations is decreased to normal levels. Considering the enzymatic parameters of MTH1 and NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role than MTH1 in preventing the occurrence of mutations that are caused by the misincorporation of 8-oxoguanine in human cells.