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

聚腺苷二磷酸核糖(PADPR)存在于所有真核生物的细胞核中 有机体。尽管多项研究表明， 这种聚合物用于修复因电离辐射等造成的损伤 目前，这种聚合物的生物学作用尚不清楚。因此， 这项研究的最终目的是确定这一现象的性质 参与修复的可能是哺乳动物细胞。要回答这个问题 问题：我们已经分离到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烷化剂异常敏感，可能还对伽马射线敏感。 为了确定这种敏感性的基础，Padr突变细胞将 检查DNA损伤的频率增加，切除中的缺陷 修复，单链和双链DNA断裂，修复复制。一个 PadR突变体姐妹染色单体交换频率的增加将 还将检查NAD和ATP池的减少。这个 突变体染色质结构改变的可能性将是 通过确定EMS诱导组蛋白聚合体的模式进行检查 二维凝胶电泳法测定ADP-核糖基化。变种人的 还将检查聚(ADP-核糖)的核基质分布 作为突变体对电穿孔敏感性的改变 限制酶。聚腺苷二磷酸核糖糖苷酶缺失突变株 将通过筛选仍有32P标记的菌落来分离 3-氨基苯甲酰胺对聚ADP-核糖合成的抑制作用突变者 DNA断裂诱导聚(ADP-核糖)合成的缺陷 将通过筛选聚(ADP-核糖)聚合酶的菌落来分离 伽马辐射后的活性和分离不起作用的菌落 合成聚合物。聚腺苷二磷酸核糖聚合酶在菌落中的活性 用32P-NAD标记和放射自显影进行检测。我们相信 聚腺苷二磷酸核糖缺陷突变株的生化特性 为阐明这种聚合物在dna中的作用提供了前景。 修理。

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.