Hahn T R, West S, Howard-Flanders P
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
J Biol Chem. 1988 May 25;263(15):7431-6.
RecA protein from Escherichia coli promotes homologous pairing and strand exchange between duplex DNA molecules if one is partially single-stranded. Using linear duplexes and circles with a single-stranded gap as the substrates, this reaction generates nicked circular heteroduplex DNA and linear molecules with single-stranded ends. The completion of strand exchange can be demonstrated by the production of nicked circular heteroduplex DNA detected by gel electrophoresis and autoradiography using radiolabeled linear molecules. When the effect of ultraviolet damage to the substrate DNA was tested, strand exchange was found to pass 30 or more pyrimidine dimers in each duplex. In contrast, exchanges were blocked or severely slowed by interstrand cross-links and monoadducts produced by psoralen and 360 nm light. Deletions and insertions of from 4 to 38 base pairs in the DNA substrates had little effect on the production of nicked circular heteroduplex DNA. However, those of 120 base pairs, or greater, reduced the product yield to a level below the threshold of detection. These results contrast with those obtained in related three-stranded reactions (Bianchi, M. E., and Radding, C. M. (1984) Cell 35, 511-520), in which stable heteroduplex products with 500 or 1300 unpaired bases were obtained when the insert was located within a single-stranded circular substrate.
如果双链DNA分子中有一条部分为单链，来自大肠杆菌的RecA蛋白会促进双链DNA分子之间的同源配对和链交换。以线性双链体和带有单链缺口的环状分子作为底物，该反应会产生带切口的环状异源双链DNA和带有单链末端的线性分子。通过使用放射性标记的线性分子进行凝胶电泳和放射自显影检测到的带切口的环状异源双链DNA的产生，可以证明链交换的完成。当测试紫外线对底物DNA的损伤作用时，发现链交换能够跨越每条双链中30个或更多的嘧啶二聚体。相比之下，补骨脂素和360nm光产生的链间交联和单加合物会阻止或严重减缓交换。DNA底物中4到38个碱基对的缺失和插入对带切口的环状异源双链DNA的产生影响不大。然而，120个碱基对或更长的缺失和插入会将产物产量降低到检测阈值以下的水平。这些结果与在相关三链反应中获得的结果形成对比（比安奇，M.E.，和拉丁，C.M.（1984年）《细胞》35卷，511 - 520页），在该反应中，当插入片段位于单链环状底物内时，会获得具有500或1300个未配对碱基的稳定异源双链产物。
