POLY (ADP-RIBOSE) AND REPAIR OF RADIATION INDUCED DAMAGE

多聚ADP核糖（pADPR）存在于所有真核生物的细胞核中， 有机体 尽管一些研究表明， 这种聚合物在修复由电离辐射和其他辐射引起的损伤中的应用 虽然这些药物的生物学作用尚不清楚。 因此 这项研究的最终目标是确定这种情况的性质， 参与修复的可能是哺乳动物细胞。 回答这个 我们已经分离出中国仓鼠卵巢细胞突变体（PADR）， 缺乏pADPR合成，并发现这些突变体也 对DNA烷化剂和可能对伽马射线异常敏感。 为了确定这种敏感性的基础，将对PADR突变体细胞进行细胞培养。 检查DNA损伤频率增加，切除缺陷 修复、单链和双链DNA断裂以及修复复制。 一个 PADR突变体中姐妹染色单体交换频率增加将 也将作为NAD和ATP池的减少进行检查。 的 突变体染色质结构改变的可能性将是 通过确定EMS诱导的组蛋白多聚体的模式进行检查， 使用二维凝胶电泳的ADP-核糖基化。 变异人的 还将检查聚（ADP-核糖）的核基质分布 作为突变体对电穿孔的敏感性的改变， 限制酶 聚（ADP-核糖）糖醛酸酶缺陷突变体 将通过筛选后仍保持32P标记的菌落进行分离 用3-氨基苯甲酰胺抑制聚（ADP-核糖）合成。 突变体 通过DNA断裂诱导聚（ADP-核糖）合成的缺陷 将通过筛选菌落的聚（ADP-核糖）聚合酶进行分离 γ-辐射后的活性，并分离不 合成聚合物。 菌落中的聚（ADP-核糖）聚合酶活性 用32P-NAD标记和放射自显影检测。 我们认为 聚（ADP-核糖）缺陷突变体的生物化学表征 为阐明这种聚合物在DNA中的作用提供了前景 修复.

Poly(ADP-ribose) (pADPR) is present in the nuclei of all eukaryotic organisms. Although a number of studies have suggested an involvement of this polymer in repair of damage produced by ionizing radiation and other agents, the biological role of the polymer remains unclear. Thus, the ultimate goal of this research is to determine what the nature of this involvement in repair might be in mammalian cells. To answer this question we have isolated Chinese hamster ovary cell mutants (PADR) that are deficient in pADPR synthesis and find that these mutants are also abnormally sensitive to DNA alkylating agents and possibly to gamma rays. To determine the basis of this sensitivity, PADR mutant cells will be examined for increased frequency of DNA damage, defects in excision repair, single and double strand DNA breaks, and repair replication. An increase in frequency in sister-chromatid exchanges in PADR mutants will also be examined as will as a reduction in NAD and ATP pools. The possibility of an alteration in the mutant's chromatin structure will be examined by determining the pattern of EMS-induced histone poly ADP-ribosylation using 2 dimensional gel electrophoresis. The mutant's nuclear matrix distribution of poly (ADP-ribose) will be examined as well as an alteration in the mutant's sensitivity to electroporated restriction enzyme. Mutants deficient in poly(ADP-ribose) glycohydolase will be isolated by screening for colonies that remain 32P-labeled after inhibition of poly(ADP-ribose) synthesis with 3-aminobenzamide. Mutants defective in the induction of poly(ADP-ribose) synthesis by DNA breaks will be isolated by screening colonies for poly(ADP-ribose) polymerase activity after gamma-irradiation and isolating colonies that do not synthesize the polymer. Poly(ADP-ribose) polymerase activity in colonies is detected using labeling with 32P-NAD and autoradiography. We believe that biochemical characterization of poly(ADP-ribose) deficient mutants offers the prospect of elucidating the role of this polymer in DNA repair.