CELLULAR RESPONSE TO DNA ADDUCTS

遗传毒剂主要通过破坏细胞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修复受损的DNA。 细胞试图在DNA复制或细胞分裂开始之前修复DNA损伤。 然而，在不期望的情况下，受损DNA的复制仍然发生。 未修复的DNA损伤通常会阻止DNA合成的进展，并且是突变的主要来源。在这个项目中，将使用内源性产生的DNA加合物，如1，N6-乙烯脱氧腺苷和丙烯醛衍生的脱氧鸟苷加合物之一，对未修复的DNA损伤的细胞反应机制进行研究。 这些加合物被怀疑有助于衰老和癌症。 由于它们在细胞DNA中连续产生，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错误的损伤耐受机制，细胞DNA的存活和完整性将仅取决于跨损伤DNA合成的效率和保真度，并且少量的阻断损伤将是致命的。 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加合物的反应是无错和易错的。 我们的初步研究表明，在E.杆菌 这种生物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阻滞。 我们已经证明了这两种途径的存在，在DNA序列，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源自各种细胞途径的子代的来源。 这种新的方法将用于探索真核生物中无错误和易出错的损伤耐受机制。 将使用人细胞和酵母中的质粒和染色体底物研究机制。 影响损伤耐受机制的因素和DNA损伤的诱导也将进行研究。

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.