CELLULAR RESPONSE TO DNA ADDUCTS

细胞对 DNA 加合物的反应

• 批准号: 6172736
• 负责人: Masaaki Moriya
• 金额: $ 17.93万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-15 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6172736
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; adduct; deoxyadenosines; electroporation; eukaryote; gene mutation; human tissue; linkage mapping; nucleic acid hybridization; nucleic acid sequence; polymerase chain reaction; transfection; western blottings; yeasts

Genotoxic agents exert their deleterious effects mainly by damaging cellular DNA. In response, cells have evolved several ways to overcome their harmful effects. One of the representative mechanisms is the repair of damaged DNA. Cells attempt to repair DNA damage before the onset of DNA replication or cell division. However, in the undesirable situation, replication of damaged DNA still occurs. Unrepaired DNA lesions often block the progression of DNA synthesis and are the major source of mutations. In this project, mechanisms for cellular responses to unrepaired DNA lesions will be studied using endogenously produced DNA adducts such as 1,N6-ethenodeoxyadenosine and one of the acrolein-derived deoxyguanosine adducts. These adducts are suspected to contribute to aging and cancer. Since they are continuously produced in cellular DNA, it is not unlikely that the cellular replication machinery encounters unrepaired endogenous lesions. If cells did not have any error-free damage tolerance mechanism, the survival and integrity of cellular DNA would depend solely on the efficiency and fidelity of translesion DNA synthesis, and a small number of blocking lesions would be lethal. However, many studies have shown that cells tolerate many unrepaired lesions. On the other hand, if cells had only error-free damage tolerance mechanism, cells would not be mutable by DNA adducts. However, cells are mutable by DNA adducts. Our central hypothesis is that cells respond to unrepaired DNA adducts in an error-free and an error-prone manner. Our preliminary studies have indicated that this is true in E. coli. This organism overcomes synthesis block by error-prone translesion synthesis and error-free daughter strand gap repair. We have demonstrated the existence of these two pathways, at the DNA sequence, using our recently developed approach. This approach utilizes a site-specifically placed single DNA adduct and strand-specific marker sequences to identify the origin of progeny which are derived from various cellular pathways. This new approach will be used to explore error-free and error-prone damage tolerance mechanisms in eukaryotes. The mechanisms will be investigated using plasmid and chromosomal substrates in human cells and yeast. The factors influencing damage tolerance mechanisms and their induction by DNA damage will also be investigated.

基因毒性物质主要通过破坏细胞DNA发挥其有害作用。作为回应，细胞已经进化出几种方法来克服它们的有害影响。其中一个代表性的机制是受损DNA的修复。在DNA复制或细胞分裂开始之前，细胞试图修复DNA损伤。然而，在不希望的情况下，受损DNA的复制仍然会发生。未修复的DNA损伤常常阻碍DNA合成的进展，是突变的主要来源。在这个项目中，细胞对未修复的DNA损伤的反应机制将使用内源性产生的DNA加合物，如1，n6 -乙烯脱氧腺苷和一种丙烯醛衍生的脱氧鸟苷加合物来研究。这些加合物被怀疑会导致衰老和癌症。由于它们在细胞DNA中不断产生，因此细胞复制机制遇到未修复的内源性损伤并非不可能。如果细胞没有任何无差错损伤耐受机制，细胞DNA的存活和完整性将完全取决于翻译DNA合成的效率和保真度，少量的阻断病变将是致命的。然而，许多研究表明，细胞耐受许多未修复的病变。另一方面，如果细胞只有无差错的损伤容忍机制，细胞就不会被DNA加合物所改变。然而，细胞是可变的DNA加合物。我们的中心假设是，细胞对未修复的DNA加合物的反应是一种无错误和易出错的方式。我们的初步研究表明，这在大肠杆菌中是正确的。该生物通过易出错的翻译合成和无差错的子链间隙修复来克服合成阻滞。我们已经证明了这两种途径的存在，在DNA序列中，使用我们最近开发的方法。该方法利用位点特异性放置的单个DNA加合物和链特异性标记序列来鉴定来自各种细胞途径的后代的起源。这种新方法将用于探索真核生物中无错误和易出错的损伤容忍机制。该机制将在人类细胞和酵母中使用质粒和染色体底物进行研究。影响损伤耐受机制的因素以及DNA损伤对损伤耐受机制的诱导也将进行研究。