HUMAN DNA-REPAIR METHYLTRANSFERASE

人类 DNA 修复甲基转移酶

• 批准号: 3460716
• 负责人: Neil D. Clarke
• 金额: $ 10.6万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3460716
• 关键词: DNA binding protein; DNA methylation; DNA repair; Escherichia coli; active sites; alanine; alkylation; chemical kinetics; crystallization; cysteine; guanine analog; high performance liquid chromatography; human genetic material tag; methyltransferase; protein purification; protein sequence; serine; site directed mutagenesis; synthetic nucleic acid; synthetic protein; thymine; transmethylation

The goal of this research is to understand how human 06-methylguanine-methyltransferase (06meG-MT) recognizes and repairs the DNA alkylation product 06-methylguanine (06meG) and the similar minor product 04-methylthymine (04meT). The repair of these modified bases by 06meG-MT helps protect cells from the mutagenic consequences of alkylation damage. The protein acts by transferring a methyl group (or other alkyl group) from DNA to itself in a suicide reaction, using a cysteine as the alkyl-acceptor. Our general approach is: (i) Express human 06meG-MT in E. coli and purify the protein. (ii) Identify mutant proteins that are defective in the recognition of methylated DNA or in the methyltransfer reaction. Mutants will be obtained by site-directed mutagenesis of the conserved, polar residues that are most likely to be functionally important. A procedure for selection of non-functional 06meG-MT mutants will also be investigated. (iii) Biochemically characterize wild-type and mutant proteins. This includes analysis of methyltransfer kinetics, measurement of the pKa for protonation of the active site cysteine, and determination of the O6meG-DNA binding properties of methyltransfer-defective mutants. (iv) Co-crystallize a methyltransfer-defective mutant of the protein with methylated DNA as a preliminary step in the determination of its structure. These studies will help in understanding how O6meG-MT reduces the mutagenic and carcinogenic effects of an important class of mutagen.

这项研究的目的是了解人类如何 06-甲基鸟嘌呤-甲基转移酶 (06meG-MT) 识别并修复 DNA烷基化产物06-甲基鸟嘌呤（06meG）和类似的次要产物 产品04-甲基胸腺嘧啶(04meT)。 这些修饰碱基的修复 06meG-MT 有助于保护细胞免受以下突变的影响 烷基化损伤。 该蛋白质通过转移甲基（或 其他烷基）在自杀反应中从 DNA 到自身，使用 半胱氨酸作为烷基受体。 我们的一般做法是： (i) 在大肠杆菌中表达人06meG-MT并纯化蛋白质。 (ii) 识别识别缺陷的突变蛋白 甲基化 DNA 或在甲基转移反应中。 突变体将是 通过保守的极性残基的定点诱变获得 这些最有可能在功能上很重要。 一个程序 还将研究非功能性 06meG-MT 突变体的选择。 (iii) 野生型和突变型蛋白质的生化特征。 这 包括甲基转移动力学分析、pKa 测量 活性位点半胱氨酸的质子化，以及测定 甲基转移缺陷突变体的 O6meG-DNA 结合特性。 (iv) 与蛋白质的甲基转移缺陷突变体共结晶 甲基化 DNA 作为测定其甲基化的初步步骤 结构。 这些研究将有助于了解 O6meG-MT 如何减少 一类重要诱变剂的诱变和致癌作用。