CELLULAR RESPONSE TO DNA ADDUCTS

遗传毒性物质主要通过破坏细胞DNA来发挥其有害作用。作为回应，细胞进化出了几种方法来克服它们的有害影响。其中具有代表性的机制之一是DNA损伤的修复。细胞试图在DNA复制或细胞分裂开始之前修复DNA损伤。然而，在不受欢迎的情况下，仍会发生受损DNA的复制。未修复的DNA损伤经常阻碍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产生的DNA加合物，如1，N6-乙叉脱氧腺苷和一种丙烯醛衍生的脱氧鸟苷加合物来研究。这些加合物被怀疑与衰老和癌症有关。由于它们在细胞DNA中不断产生，细胞复制机制遇到无法修复的内源性损伤并不是不可能的。如果细胞没有任何无错误的损伤耐受机制，细胞DNA的存活和完整性将完全取决于跨损伤DNA合成的效率和保真度，少量的阻断损伤将是致命的。然而，许多研究表明，细胞可以耐受许多未修复的损伤。另一方面，如果细胞只有无错误的损伤耐受机制，细胞就不会被DNA加合物突变。然而，细胞通过DNA加合物是可变的。我们的中心假设是，细胞对未修复的DNA加合物以一种没有错误和容易出错的方式做出反应。我们的初步研究表明，在大肠杆菌中也是如此。这种有机体通过容易出错的跨损伤合成和无错误的子链缺口修复来克服合成障碍。我们已经用我们最近开发的方法在DNA序列上证明了这两条途径的存在。这种方法利用位置特异的单DNA加合物和链特异的标记序列来鉴定来自不同细胞途径的后代的起源。这一新的方法将被用来探索真核生物中无错误和容易出错的损伤容忍机制。这一机制将利用人类细胞和酵母中的质粒和染色体底物进行研究。还将探讨影响损伤耐受机制的因素以及DNA损伤对其的诱导作用。

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.