Abstract
Nucleotides in the free pool are more susceptible to nonenzymatic methylation than those protected in the DNA double helix. Methylated nucleotides like O6-methyl-dGTP can be mutagenic and toxic if incorporated into DNA. Removal of methylated nucleotides from the nucleotide pool may therefore be important to maintain genome integrity. We show that MutT homologue 1 (MTH1) efficiently catalyzes the hydrolysis of O6-methyl-dGTP with a catalytic efficiency similar to that for 8-oxo-dGTP. O6-methyl-dGTP activity is exclusive to MTH1 among human NUDIX proteins and conserved through evolution but not found in bacterial MutT. We present a high resolution crystal structure of human and zebrafish MTH1 in complex with O6-methyl-dGMP. By microinjecting fertilized zebrafish eggs with O6-methyl-dGTP and inhibiting MTH1 we demonstrate that survival is dependent on active MTH1 in vivo. O6-methyl-dG levels are higher in DNA extracted from zebrafish embryos microinjected with O6-methyl-dGTP and inhibition of O6-methylguanine-DNA methyl transferase (MGMT) increases the toxicity of O6-methyl-dGTP demonstrating that O6-methyl-dGTP is incorporated into DNA. MTH1 deficiency sensitizes human cells to the alkylating agent Temozolomide, a sensitization that is more pronounced upon MGMT inhibition. These results expand the cellular MTH1 function and suggests MTH1 also is important for removal of methylated nucleotides from the nucleotide pool.
Highlights
DNA can be methylated nonenzymatically by environmental methylating agents, chemotherapeutics such as Temozolomide [1] and natural cellular methyl donors like Sadenosylmethionine (SAM) [2]
Comparison of activities of MutT homologue 1 (MTH1) with O6-methyl-dGTP, N2-methyl-dGTP, dGTP and the previously best known MTH1 substrate 8oxo-dGTP showed that O6-methyl-dGTP was hydrolyzed at a level similar to that of 8-oxo-dGTP (Figure 1A)
The MTH1 catalyzed hydrolysis of O6methyl-dGTP was analysed using HPLC and LC–mass spectrometry (MS) showing that the product formed is O6-methyl-dGMP as expected (Supplementary Figure S1A)
Summary
DNA can be methylated nonenzymatically by environmental methylating agents, chemotherapeutics such as Temozolomide [1] and natural cellular methyl donors like Sadenosylmethionine (SAM) [2]. Free nucleotides are reported to be ∼190–13 000 times more susceptible to methylation damage, depending on the site of methylation, compared to when present in the DNA [3]. This is likely due to a higher accessibility of free nucleotides compared to when present in the densely packed DNA. The order of susceptibility for methylation of free nucleotides by N-methyl-Nnitrosourea was found to be dependent on the site on the base, with methylations at the N1 position of adenine being the most common, followed by the N3 position of adenine. N3-methyl-A blocks replication, O6-methyl-G induces G:C to A:T mutations [4] and causes DNA strand breaks and replication fork collapse [5] and N7-methyl-G gives rise to mutagenic apurinic sites and blocks DNA replication [6,7,8]
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