CELLULAR PROCESSING OF DAMAGED DNA: ROLE IN ONCOGENESIS

我们的最终目标是了解如何处理 哺乳动物细胞中的DNA损伤与癌症的发生有关。 使用相对较短的、定义的DNA序列，包含Well 特征损伤，我们已经开始分析基因组内 培养细胞DNA修复的“精细结构”。拥有 发现核基因组的某些区域修复了 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取决于 病变类型、其在基因组中的位置和功能状态 病变部位的DNA。这种专一性可能 解释了即将出现的一些深刻差异 不同组织和同一组织的致癌反应 在不同的生物体中。已经开发出足够灵敏的分析方法 检测几种不同损伤的修复情况，包括嘧啶 二聚体和链间交链，在来自 基因组的特定区域，我们将比较它们的速率和 不同表达水平的基因的修复程度，时间 复制、基因组位置和功能。示例包括 原癌基因和其他可诱导或发育激活的基因 金属硫蛋白、甲胎蛋白、胎儿 与成体β-珠蛋白和肌球蛋白重链的分化 成肌细胞。染色质构象和甲基化水平将 被评估为熟练维修的可能决定因素。修补 而突变将在相同的基因中相互关联 确定差异修复是否可能解释 与致癌相关的诱变变化。复制 已定义的含有损伤的核苷酸序列将被研究以 确定中是否存在差异级别的复制 特定的基因组结构域和子链是否 在这些序列中会出现不连续。定义嵌合体 含有独特部位损伤的质粒将被用来 将基因导入不同的基因组结构域并探索 损伤处理的特定特征增加了频率 人类细胞的稳定转化。 这项研究应该对我们的理解有很大的帮助 DNA损伤处理缺陷的基础在某些方面 易患癌症的遗传性疾病，它还应该导致新的， 用于人体损伤与修复分析的灵敏探针 细胞。此外，我们的研究应该有助于解释 生物终点的DNA损伤，如存活、突变、 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.