An O6-methylguanine-DNA methyltransferase-like protein from Thermus thermophilus interacts with a nucleotide excision repair protein
An O6-methylguanine-DNA methyltransferase-like protein from Thermus thermophilus interacts with a nucleotide excision repair protein
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DOI：
10.1093/jb/mvn065
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发表时间：
2008-08-01
影响因子：
2.7
通讯作者：
Masui, Ryoji
中科院分区：
文献类型：
--
作者：
Morita, Rihito;Nakagawa, Noriko;Masui, Ryoji
烷化剂对DNA的主要损伤包括O-6-甲基鸟嘌呤（O-6-meG）的形成。几乎所有物种都具有O-6-甲基鸟嘌呤-DNA-甲基转移酶（Ogt）来修复这种损伤。Ogt通过甲基化学计量转移到其活性位点（PCHR）的半胱氨酸残基来修复DNA中的O-6-meG损伤。嗜热栖热菌HB 8有一个Ogt同源物TTHA 1564，但在这种情况下，丙氨酸残基取代半胱氨酸在推定的活性位点。为了揭示TTHA 1564在处理含O-6-meG的DNA中的可能功能，我们表征了TTHA 1564的生化性质。TTHA 1564对合成的含O-6-meG的DNA没有甲基转移酶活性，表明TTHA 1564是一种烷基转移酶样蛋白。然而，凝胶迁移分析表明TTHA 1564可以以比正常（未甲基化）DNA更高的亲和力（9倍）结合含有O-6-meG的DNA。使用荧光寡核苷酸的实验表明TTHA 1564使用与其他Ogts相同的机制识别DNA中的O-6-meG。然后，我们研究了TTHA 1564是否作为损伤传感器。下拉分析鉴定了20种蛋白质，包括核苷酸切除修复蛋白UvrA，其与TTHA 1564相互作用。TTHA 1564与UvrA的相互作用使用表面等离子体共振测定来证实。这些结果表明TTHA 1564可能参与DNA修复途径。
The major damage to DNA caused by alkylating agents involves the formation of O-6-methylguanine (O-6-meG). Almost all species possess O-6-methylguanine-DNA-methyltransferase (Ogt) to repair such damage. Ogt repairs O-6-meG lesions in DNA by stoichiometric transfer of the methyl group to a cysteine residue in its active site (PCHR). Thermus thermophilus HB8 has an Ogt homologue, TTHA1564, but in this case an alanine residue replaces cysteine in the putative active site. To reveal the possible function of TTHA1564 in processing O-6-meG-containing DNA, we characterized the biochemical properties of TTHA1564. No methyltransferase activity for synthetic O-6-meG-containing DNA could be detected, indicating TTHA1564 is an alkyltransferase-like protein. Nevertheless, gel shift assays showed that TTHA1564 can bind to DNA containing O-6-meG with higher affinity (9-fold) than normal (unmethylated) DNA. Experiments using a fluorescent oligonucleotide suggested that TTHA1564 recognizes O-6-meG in DNA using the same mechanism as other Ogts. We then investigated whether TTHA1564 functions as a damage sensor. Pull-down assays identified 20 proteins, including a nucleotide excision repair protein UvrA, which interacts with TTHA1564. Interaction of TTHA1564 with UvrA was confirmed using a surface plasmon resonance assay. These results suggest the possible involvement of TTHA1564 in DNA repair pathways.