CELLULAR PROCESSING OF DAMAGED DNA: ROLE IN ONCOGENESIS

我们的最终目标是了解 哺乳动物细胞中受损的DNA与致癌作用有关。 使用相对较短的、定义明确的DNA序列， 特征性病变，我们已经开始分析基因组内 培养细胞中DNA修复的“精细结构”。 具有 发现细胞核基因组的某些区域 比其他人更有效率，我们假设效率 哺乳动物染色质损伤的修复依赖于 病变类型、其在基因组中的位置和功能状态 在病变部位的DNA。 这种特异性可以 解释了一些深刻的差异很快在 不同组织和同一组织的致癌反应 在不同的生物体中。 开发出足够灵敏的检测方法 检测几种不同损伤的修复，包括嘧啶 二聚体和链间交联，在限制性片段中， 基因组的特定区域，我们将比较率和 不同表达水平的基因的修复程度， 复制、基因组定位和功能。 实例包括 原癌基因和其他可诱导的或发育激活的 例如金属硫蛋白基因、甲胎蛋白基因、胎儿 和成人β-珠蛋白和肌球蛋白重链在分化 成肌细胞 染色质构象和甲基化水平将 被评估为熟练修复的可能决定因素。 修复 而突变将在相同的基因中与 确定差异修复是否可以解释 与致癌作用有关的诱变变化。 复制 将研究含有损伤的确定的核苷酸序列， 确定是否发生不同级别的复制 特定的基因组结构域以及子链是否 在这些序列中出现不连续性。 确定的嵌合 在独特位点含有损伤的质粒将用于 将基因引入不同的基因组结构域， 具体损伤特征处理频率增加 人类细胞的稳定转化。 这项研究将大大有助于我们理解 DNA损伤处理缺陷的基础， 易患癌症的遗传性疾病，它也会导致新的， 用于分析人体损伤和修复的灵敏探针 细胞 此外，我们的研究应该有助于解释的作用， 生物学终点中的DNA损伤，如存活、诱变， 和致癌作用。

Our ultimate objective is to understand how the processing of damaged DNA in mammalian cells relates to carcinogenesis. Using relatively short, defined DNA sequences containing well characterized lesions, we have begun to analyze the intragenomic "fine structure" of DNA repair in cultured cells. Having discovered that certain regions of the nuclear genome repaired more efficiently than others, we hypothesize that the efficiency of repair of damage in mammalian chromatin depends upon the type of lesion, its location in the genome and the functional state of the DNA at the site of the lesion. Such specificity may account for some of the profound differences soon in the carcinogenic responses of different tissues and of the same tissue in different organisms. Having developed assays sensitive enough to detect repair of several different lesions, including pyrimidine dimers and interstrand cross-links, in restriction fragments from specific regions of the genome, we will compare the rate and extent of repair in genes that differ levels of expression, time of replication, genomic location and function. Examples include protooncogenes and other inducible or developmentally activated genes such as those for metallothioneins, alpha fetoprotein, fetal and adult beta-globin and myosin heavy chain in differentiating myoblasts. Chromatin conformation and methylation levels will be assessed as possible determinants of proficient repair. Repair and mutagenesis will be correlated in the same genes to determine whether differential repair might account for mutagenic changes related to carcinogenesis. Replication of defined nucleotide sequences containing damage will be studied to determine whether differential levels of replication occur in particular genomic domains and whether daughter-strand discontinuities occur in those sequences. Defined chimeric plasmids containing lesions at unique sites will be used to introduce genes into different genomic domains and to probe the specific features of damage processing the increase the frequency of stable transformation of human cells. This research should contribute substantially to our understanding of the basis for DNA damage processing deficiencies in certain cancer-prone hereditary diseases and it should also result in new, sensitive probes for the analysis of damage and repair in human cells. In addition, our studies should help to interpret the role of DNA damage in biologic end points such as survival, mutagenesis, and carcinogenesis.