Molecular genetics and structural biology of human MutT homolog, MTH1

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Abstract

The human MTH1 gene located on chromosome 7p22 consists of 5 major exons. MTH1 gene produces seven types of mRNAs and the B-type mRNAs with exon 2b–2c segments direct synthesis of three forms of MTH1 polypeptides (p22, p21, and p18) by alternative initiation of translation, while the others encode only p18. In human cells, p18, the major form is mostly localized in the cytoplasm with some in the mitochondria. A single nucleotide polymorphism (SNP) in exon 2, which is tightly liked to another SNP (GTG83/ATG83), creates an additional alternative in-frame AUG in B-type MTH1 mRNAs yielding the fourth MTH1 polypeptide, p26 that possesses an additional mitochondrial targeting signal. These SNPs are likely to be one of the risk factors for cancer or for neuronal degeneration. The 30 amino acid residues are identical between MTH1 and MutT, and there is a highly conserved region consisting of 23 residues (MTH1: Gly36 to Gly58), with 14 identical residues. A chimeric protein in which the 23 residue sequence of MTH1 was replaced with that of MutT, retains the capability to hydrolyze 8-oxo-dGTP, indicating that the 23 residue sequences of MTH1 and MutT are functionally and structurally equivalent, and constitute a functional phosphohydrolase module. Saturated mutagenesis of the module in MTH1 indicated that an amphipathic property of the α-helix I consisting of 14 residues of the module (Thr44 to Gly58) is essential to maintain the stable catalytic surface for 8-oxo-dGTPase. MTH1 but not MutT efficiently hydrolyzes two forms of oxidized dATP, 2-hydroxy-dATP and 8-oxo-dATP, as well as 8-oxo-dGTP and 8-oxo-GTP. Thus, MTH1 is designated as the oxidized purine nucleoside triphosphatase and has a much wider substrate specificity than MutT. There is a significant homology between MTH1 protein and the C-terminal half of human MYH protein, which may be involved in the recognition of 8-oxoguanine and 2-hydroxyadenine.

Introduction

Endogenous oxidation of DNA and DNA precursors by reactive oxygen species (ROS) appears to induce spontaneous mutations, aging, and various diseases, including cancer and neuronal degeneration [1], [2]. It has been established that 8-oxo-dGTP, an oxidized form of dGTP is highly mutagenic and one of main endogenous sources for spontaneous mutagenesis. During DNA replication, 8-oxo-dGTP can be inserted into the nascent strand opposite adenine and cytosine in the template, with almost equal efficiency, an event which leads to an A:T to C:G transversion mutation [3], [4], [5].

To eliminate such deleterious oxidized nucleotides, organisms come equipped with elaborate mechanisms. In Escherichia coli, the MutT protein hydrolyzes 8-oxo-dGTP to the monophosphate form, thus, eliminating mutagenic substrates from the DNA precursor pool [3]. Lack of the mutT gene increases the spontaneous occurrence of A:T to C:G transversion a 1000-fold over the wild-type level [6], [7], [8]. Moreover, it has been shown that E. coli RNA polymerase misinserts 8-oxo-GTP, an oxidized form of GTP, into mRNA, yielding mutant forms of proteins known as non-genomic mutations in mutT deficient cells. MutT protein also efficiently hydrolyzes 8-oxo-GTP, and, thus, minimizes errors caused by misincorporation of oxidized guanine nucleotides into RNA [9].

Enzymatic activity similar to that of MutT protein has been identified in human cells [10], [11]. Based on partial amino acid sequences obtained from a purified preparation of human 8-oxo-dGTPase, cDNA and the gene for the human enzyme were isolated, and named MTH1 (mutT homolog-1) [11], [12]. Since 30 amino acid residues (23%) are identical between MutT and MTH1 proteins, and expression of the human enzyme in mutT-E. coli cells suppresses the elevated level of spontaneous mutation frequency to an almost normal, the human enzyme appears to have the same antimutagenic capacity as does E. coli MutT protein.

Findings with regard to the human MTH1 protein, the function of which is likely to differ from those in bacteria, are reviewed herein.

Section snippets

Regulation of expression of the MTH1 gene

In human tissues, large amounts of MTH1 mRNA are present in normal thymus, testis and embryonic tissues. In peripheral blood lymphocytes (PBL), the level of MTH1 mRNA was significantly increased after concomitant treatment with phytohemagglutinin (PHA) and interleukin-2 (IL-2), indicating that expression of the MTH1 gene is inducible in human cells during proliferative activation [13].

Cancerous tissues in humans have significantly higher levels of MTH1 transcripts [14], [15], presumably the

MutT family proteins with the phosphohydrolase module

Genes for MutT homolog proteins with dGTPase or 8-oxo-dGTPase activity were identified in Proteus vulgaris and Streptococcus pneumoniae, bacteria distantly related to E. coli (Fig. 3) [7], [20], [21]. All three MutT homolog proteins have a molecular mass ranging from 13 to 18 kDa, and the degree of identity ranges from 19 to 40%. Most of the identical residues are in a region corresponding to the 23 residues from Gly37 to Gly59 of E. coli MutT, known as the MutT signature [22]. Homologs of human

Molecular epidemiology of MTH1 gene

To date, two different SNPs in human MTH1 gene, which affect expression or function of MTH1 protein, were found, as discussed above [13], [17], [32], [43]. We examined the distribution of one of the SNPs, namely Val83/Met83 in the Japanese population, living in Japan, including 400 healthy volunteers and 601 patients with various diseases. Allele frequencies of Val83 and Met83 in the healthy volunteers are 0.91 and 0.09, respectively. There were three homozygotes for Met83/Met83 out of 400

Conclusion

As summarized in Fig. 6, oxidized purine nucleoside triphosphates, such as 2-OH-dATP, 8-oxo-dGTP, 8-oxo-dATP and 8-oxo-GTP are hydrolyzed to the corresponding monophosphates by the action of MTH1 protein. It has been demonstrated that guanylate kinase, which phosphorylates GMP and dGMP to the corresponding nucleoside diphosphates is inactive on 8-oxo-dGMP, thus, preventing re-utilization of the MTH1 cleavage product during DNA replication or transcription [44]. 8-Oxo-dGMP is rapidly

Acknowledgements

I extend special thanks to Drs. M. Furuichi, K. Sakumi, Y. Sakai, T. Tsuzuki, M. Takahashi, M. Shirakawa, and M. Sekiguchi, for helpful discussions, and to M. Ohara for language assistance.

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