INTERSTRAND CROSSLINKS--REPLICATION, REPAIR, MUTATIONS

链间交联——复制、修复、突变

• 批准号: 2093199
• 负责人: EDWARD L LOECHLER
• 金额: $ 14.09万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2093199
• 关键词: DNA damage; DNA repair; DNA replication; Escherichia coli; adduct; alkylating agents; antineoplastics; crosslink; deToni Fanconi syndrome; genetic recombination; genome; kidney cell; nitrogen mustard; oligonucleotides; site directed mutagenesis; tissue /cell culture; transfection /expression vector; xeroderma pigmentosum

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.

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， 它是由双功能活性化合物形成的，如氮 芥末、双氯乙基亚硝脲和丝裂霉素C。 细胞毒性，是诱变的，并通过以下机制在细胞中修复 不是很清楚。使用重组DNA和化学物质的组合 合成技术、方法被开发出来建造人类航天飞机 在确定的基因组位置包含链间交叉链接的载体 使用现场定向方法。这些向量将被用于研究 链间交联在体内和体外的生物学后果。 这一点很重要，因为交联剂经常被使用 在治疗癌症方面。 构建的第一个载体(指定为interhn2-pTZSV28)包含 氮芥末链间交联剂。在E. 已经在人类293(肾脏)细胞中进行了初步研究 完成。InterHN2-pTZSV28获得了较高的后代产量 与相同的载体相比，在大肠杆菌中的载体(~30%) 不含交联链。高产量被证明依赖于 切除修复，但不是重组修复。这一结果意味着 氮芥链间的交联可以通过一种 不同于通常被接受的链间路径 交叉链接修复，包括重组步骤。这是一项新的 该途径是准确的，因为来自间隔型HN2-pTZSV28的后代被证明是 成为压倒性的野性类型。在初步研究中-hn2-pTZSV28 在人293细胞中也获得了高产量的子代(~60%)，并且数据 也暗示了修复途径的存在并不依赖于 重组修复。 建议进行更多的实验来证实这种现象的存在。 在大肠杆菌中修复链间交联链的第二条途径。如果 确认后，体内和体外的实验将被指导 以确定几条拟议的路径中哪一条可能是可行的。 类似的研究将在人类细胞系中进行，包括在 被认为在链间交联修复中有缺陷的细胞(例如， Fanconi‘s贫血细胞)。将对后代进行分析，以确定 突变是在原始基因组位置或附近诱导的 交叉链和频率。类似的实验将会进行。 从其他试剂衍生的链间交联链上。的存在 如果确认，链间交链修复的第二条途径可以是 与交联剂发挥作用的机制有关 细胞毒性。