Mechanisms of mutagenesis and maintenance of genetic information in mammalian cells

对于生物体来说，通过精确的DNA复制和对受损DNA的有效修复来保持其遗传信息的完整性是很重要的。我们研究了这些过程背后的分子机制。抑制诱导突变发生的蛋白编码基因的分子克隆和蛋白作用的阐明：我们克隆了人、小鼠、大鼠和兔O^6-甲基鸟嘌呤-DNA甲基转移酶的cDNA，并测定了它们的核苷酸序列。通过引入氨基酸取代，我们发现了酶活性部位附近的保守氨基酸序列。我们只检测到该基因在某些细胞系中的表达水平非常低，这些细胞系通常是从肿瘤建立的。在…的某些步骤中，一定存在其他方面的异常更多的基因表达，因为我们可以证明，基因转录的启动在缺陷细胞中发生在与正常细胞相同的水平。我们克隆了小鼠的基因组序列，并构建了靶向载体，通过制造缺陷小鼠来阐明该酶在肿瘤发生中的作用。保持DNA复制高保真的机制：要了解自发突变的控制机制，我们需要完全了解获得高保真DNA复制的分子机制。我们的研究重点是在细菌中发现的蛋白质，这些蛋白质负责将突变频率保持在非常低的水平。我们发现，人细胞提取物中含有类似于E.coliMutt蛋白的8-oxo-dGTP酶活性。我们纯化了该蛋白，测定了该蛋白的部分氨基酸序列，并用RT-PCR方法克隆了人的基因，并对相关生物的基因组和基因序列进行了克隆和分析。较少

It is important for organisms to keep their genetic information intact through generations, and this is accomplished by accurate DNA replication and efficient repair of damaged DNA.We investigated the molecular mechanisms underlying these processes.1. Molecular cloning of the genes encoding the proteins that suppress the occurrence of induced mutations and elucidation of the role of the protein : We have cloned the cDNAs of human, mouse, rat and rabbit O^6-methylguanine-DNA methyltransferases, which play a central role in repair of DNA methylated by alkylating agents and in preventing the occurrence of mutations and formation of tumors, and determined their nucleotide sequences. We found the conserved amino acid sequences around the active sites of the enzymes by introducing the amino acid substitutions. We have detected only a very low level of expression of the gene in certain cell lines, which are often established from tumors. There must be an abnormality elsewhere in some steps of … More gene expression since we could demonstrate that initiation of transcription of the gene occurs at the same level in the defective cells as in the normal cells. We have cloned the mouse genomic sequences and constructed targeting vectors for elucidation of role of the enzyme in tumorigenesis by making defective mice.2. Mechanisms of keeping high fidelity of DNA replication : To understand the controlling mechanisms for spontaneous mutagenesis we require the complete knowledge of the molecular mechanisms of attaining the high fidelity of DNA replication. We focused our studies to proteins found in bacteria to be responsible for keeping the mutation frequency at very low level. We found that human cell extracts contain an activity of 8-oxo-dGTPase similar to E.coli MutT protein. We purified the protein and determined the partial amino acid sequences of the protein and cloned human cDNA by RT-PCR.Cloning and analysis of the cDNA and genomic sequences of relevant organisms were carried out. Less