ALTERED DNA LIGASE I IN CANCER-PRONE HEREDITARY DISEASE

布鲁姆综合征患者的细胞，一种常染色体 与癌症频率增加相关的遗传性疾病， 显示出染色体异常。其中包括增加的 自发姐妹染色单体互换率(SCE)和 染色体异常；伴随着缓慢的复制子分支 DNA链成熟的进展和减慢的速度；以及 对DNA损伤剂的敏感性略有增加。其中几个 这些特征也是大肠杆菌和酵母突变株的特征。 DNA连接酶有缺陷。 我们最近发现BS的DNA连接酶I活性存在缺陷 细胞，而DNA连接酶II，DNA聚合酶的活性- α和β没有改变。不能正确地结扎 复制、修复或重组过程中的DNA断裂可能 解释了BS的许多障碍，包括严重的 增加罹患癌症的风险。 这项提案的目的是：1)确定是否减少了 酶的活性与蛋白质分子的减少有关 在BS细胞中，使用免疫印迹、连接酶-AM(32P)加合物试验，以及 SDS-凝胶电泳法；2)纯化酶，比较 正常和异常细胞的结构和生化特性 连接酶I，提高抗体，并鉴定改变的结构域；3) 筛选其他易患癌症的细胞、突变体、正常BS细胞杂交体， 以及肿瘤前组织中连接酶的异常；最后， 4)克隆正常和异常的连接酶I基因，并纠正 显微注射连接酶蛋白和/或转染法修复BS缺陷 连接酶基因。 癌症易感基因的分子本质的阐明 疾病很可能为我们提供新的洞察力 DNA加工与演绎中的必修步骤(S) 人类癌症的开端。

Cells from patients with Bloom's syndrome (BS), an autosomal inherited disorder associated with increased cancer frequency, exhibit chromosomal abnormalities. These include an increased rate of spontaneous sister chromatid exchanges (SCE) and chromosomal aberrations; together with slow replicon fork progression and a retarded rate of DNA chain maturation; and slightly increased sensitivity to DNA damaging agents. Several of these features are also characteristic of E. coli and yeast mutants with a defective DNA ligase. We recently found that DNA ligase I activity was defective in BS cells, whereas the activities of DNA ligase II, DNA polymerase- alpha and -beta were not altered. The inability to properly ligate DNA breaks during replication, repair, or recombination may account for the many disorders of BS, including the greatly increased risk of cancer. The aim of this proposal is: 1) to determine if the decrease in enzyme activity correlates with the decrease in protein molecules in BS cells using immunoblot, ligase-AM(32P) adduct assay, and SDS-gel electrophoresis; 2) to purify the enzymes, compare the structural and biochemical properties of normal and aberrant ligase I, raise antibodies, and to identify the altered domain; 3) to screen other cancer-prone cells, mutants, normal-BS cell hybrids, and pre-neoplastic tissues for abnormalities in ligases; and finally, 4) to clone the normal and aberrant ligase I genes, and correct the BS defect by microinjection of ligase protein and/or transfection of the ligase gene. The elucidation of the molecular nature of cancer-prone genetic diseases may well provide new insights into the mechanism of DNA processing and deduction of the obligatory step(s) in the initiation of human cancer.