Sladek F M, Munn M M, Rupp W D, Howard-Flanders P
Department of Molecular Biophysics, Yale University, New Haven, Connecticut 06511.
J Biol Chem. 1989 Apr 25;264(12):6755-65.
Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.
补骨脂素会产生DNA链间交联，人们认为在大肠杆菌中这种交联是通过一种连续的切除和重组机制来修复的。UvrABC进行的第一轮切割已得到表征：它会产生与完整DNA链交联的11碱基寡核苷酸（范·霍滕，B.，甘珀，B.，霍尔布鲁克，S.R.，赫斯特，J.E.，以及桑卡尔，A.（1986年）《美国国家科学院院刊》83卷，8077 - 8081页）。在本研究中，构建了在特定位置含有4'-羟甲基 - 4,5',8 - 三甲基补骨脂素（HMT）交联的DNA底物，并用于分析修复的其他步骤。结果表明，RecA蛋白介导链转移越过与单链DNA环交联的寡核苷酸，并且由此产生的异源双链体是UvrABC复合物的底物：它切除包含HMT交联的双链寡核苷酸。还发现UvrABC的第一轮切割不会直接导致链交换，而是需要一个中间步骤。该步骤在体外由DNA聚合酶I（pol I）的5'-外切核酸酶活性执行，该活性在切割的交联处产生一个单链DNA区域（一个缺口），使得RecA能够启动链交换。使用交联寡核苷酸的研究表明，pol I产生的缺口是由于聚合酶无法在距离HMT交联呋喃侧两到三个核苷酸的3'-OH末端添加核苷酸。然而，pol I可以在交联吡喃侧旁边延伸3'-OH末端。由于UvrABC主要在双链DNA中切割补骨脂素交联的呋喃侧，这种差异对修复具有重要影响。
