Pu W T, Kahn R, Munn M M, Rupp W D
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Biol Chem. 1989 Dec 5;264(34):20697-704.
The Escherichia coli UvrABC endonuclease is a multisubunit enzyme that initiates the repair of a wide variety of DNA lesions in vivo by making dual incisions on a damaged strand at the eighth or ninth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified base. It has been hypothesized that UvrABC is able to recognize a broad spectrum of lesions because it does not recognize the lesion per se but rather gross helical distortions that the lesion induces in the DNA. Several lesions have recently been studied which are thermal stabilizing and are not believed to distort the DNA grossly, including the CC-1065-N-3-adenine and anthramycin-N-2-guanine adducts. We have studied the activity of UvrABC in vitro on another thermal stabilizing and nondistortive adduct, N-methylmitomycin A (NMA), a bifunctional DNA-alkylating agent that reacts with guanine on the side facing the minor groove, yielding either monoadducts or interstrand cross-links. NMA adducts increase the thermal stability of DNA, and theoretical calculations indicate that NMA adducts do not grossly distort the DNA helix. Our results show that UvrABC makes incisions at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to an NMA monoadduct, consistent with the incision pattern observed for the majority of other lesions that are also recognized by UvrABC. DNA containing a site-specific NMA cross-link was also recognized and incised by UvrABC. The rate of incision of NMA cross-linked DNA was about 200-fold higher in supercoiled molecules than in relaxed molecules, whereas the rate of incision of DNA containing NMA monoadducts was stimulated approximately 2-fold by supercoiling. The signal for UvrABC recognition and incision of damaged DNA is discussed in relation to the ability of UvrABC to incise NMA adducts as well as other nondistortive lesions.
大肠杆菌UvrABC核酸内切酶是一种多亚基酶，通过在受损链上修饰碱基的5'端第八或第九个磷酸二酯键以及3'端第四个或第五个磷酸二酯键处进行双重切割，在体内启动对多种DNA损伤的修复。据推测，UvrABC能够识别广泛的损伤，因为它本身并不识别损伤，而是识别损伤在DNA中引起的总体螺旋扭曲。最近研究了几种具有热稳定性且据信不会严重扭曲DNA的损伤，包括CC - 1065 - N - 3 -腺嘌呤和氨茴霉素 - N - 2 -鸟嘌呤加合物。我们在体外研究了UvrABC对另一种具有热稳定性且无扭曲作用的加合物N -甲基丝裂霉素A（NMA）的活性，NMA是一种双功能DNA烷基化剂，它在面向小沟的一侧与鸟嘌呤反应，产生单加合物或链间交联。NMA加合物可提高DNA的热稳定性，理论计算表明NMA加合物不会严重扭曲DNA螺旋。我们的结果表明，UvrABC在NMA单加合物的5'端第八个磷酸二酯键和3'端第五个磷酸二酯键处进行切割，这与UvrABC识别的大多数其他损伤所观察到的切割模式一致。含有位点特异性NMA交联的DNA也被UvrABC识别并切割。在超螺旋分子中，NMA交联DNA的切割速率比松弛分子中高约200倍，而含有NMA单加合物的DNA的切割速率因超螺旋作用而提高约2倍。结合UvrABC切割NMA加合物以及其他无扭曲损伤的能力，讨论了UvrABC识别和切割受损DNA的信号。
