INTERSTRAND CROSSLINKS--REPLICATION, REPAIR, MUTATIONS

许多化学物质破坏DNA并形成各种DNA加合物。 一 DNA损伤的重要类别是链间，DNA-DNA交联， 其由双官能反应性化合物形成，例如氮 芥子气、双-氯乙基亚硝基脲和丝裂霉素C。 这些化学物质 细胞毒性，是诱变性的，并在细胞中通过 没有很好地理解。 使用重组DNA和化学物质的组合 合成技术，方法被开发来构建人类穿梭机 在确定的基因组位置含有链间交联的载体 使用定点方法。 这些载体将用于研究 体内和体外链间交联的生物学后果。 这是重要的，因为交联剂经常使用 在癌症治疗中的应用 构建的第一个载体（命名为inter-HN 2-pTHBV 28）含有 氮芥股间交联。 本文对E. 在人293（肾）细胞中的初步研究已经被 完成 Inter-HN 2-pTHBV 28产生了相对高的后代产量 E.大肠杆菌中的表达， 不含交联。 高产率取决于 切除修复，而不是重组修复。 这一结果意味着 氮芥链间交联可以通过 除了通常被接受的链间途径之外， 交联修复，其包括重组步骤。 这个新 途径是准确的，因为来自HN 2-pTZSV 28间的后代被证明 绝对是野生型的 在初步研究中，inter-HN 2-pTHBV 28 在人293细胞中也产生了高产率的后代（~60%），并且数据 还表明，修复途径的存在，不依赖于 重组修复 提出了额外的实验来证实这一点的存在。 修复E.杆菌 如果 证实，然后在体内和体外实验将直接 以确定几个拟议的途径中的哪一个可能是有效的。 类似的研究将在人类细胞系中进行，包括那些 认为在链间交联修复中有缺陷的细胞（例如， Fanconi贫血细胞）。 将分析后代以确定是否 突变是在或接近原始基因组位置诱导的， 交叉连接以及频率。 将进行类似的实验 来自其他试剂的链间交联。 的存在 如果得到证实，则可以使用第二种链间交联修复途径， 与交联剂发挥其作用的机制相关 细胞毒性。

Many chemicals damage DNA and form a variety of DNA adducts. One important class of DNA damage is the interstrand, DNA-DNA cross-link, which is formed by bifunctionally reactive compounds, such as nitrogen mustard, bis-chloroethylnitrosourea and mitomycin C. These chemicals are cytotoxic, are mutagenic and are repaired in cells by mechanisms that are not well understood. Using a combination of recombinant DNA and chemical synthetic techniques, methods were developed to construct human shuttle vectors that contain interstrand cross-links at defined genome locations using site-directed methods. These vectors will be used to study the biological consequences of interstrand cross-links in vivo and in vitro. This is of significance because cross-linking agents are frequently used in the treatment of cancer. The first vector constructed (designated, inter-HN2-pTZSV28) contained a nitrogen mustard interstrand cross-link. A series of studies in E. coli and preliminary studies in human 293 (kidney) cells have been completed. Inter-HN2-pTZSV28 gave a relatively high yield of progeny vectors (~30%) in E. coli in comparison to an identical vector that contained no cross-link. the high yield was shown to depend upon excision repair, but not recombinational repair. This result implied that the nitrogen mustard interstrand cross-link can be repaired by a pathway other than the one that is commonly accepted for interstrand cross-link repair, which includes a recombinational step. This new pathway is accurate in that progeny from inter-HN2-pTZSV28 were shown to be overwhelmingly wild type. In preliminary studies inter-HN2-pTZSV28 also gave a high yield of progeny (~60%) in human 293 cells, and the data also suggested that a repair pathway exists that does not depend upon recombinational repair. Additional experiments are proposed to confirm the existence of this second pathway for the repair of interstrand cross-links in E. coli. If confirmed, then experiments both in vivo and in vitro will be directed toward determining which of several proposed pathways might be operative. Similar studies will be performed in human cell lines, including in those cells thought to be defective in interstrand cross-link repair (e.g., Fanconi's anemia cells). Progeny will be analyzed to determine whether mutations are induced at or near the original genome location of the cross-link and at what frequency. Similar experiments will be conducted on interstrand cross-links derived from other agents. the existence of a second pathway for interstrand cross-link repair, if confirmed, may be of relevance to the mechanism by which cross-linking agents exert their cytotoxic.