Formation and enzymatic properties of the UvrB.DNA complex.
Formation and enzymatic properties of the UvrB.DNA complex.
复制标题
DOI：
--
复制
发表时间：
1990-09
期刊：
影响因子：
--
通讯作者：
David K. Orren;Aziz Sancar
中科院分区：
文献类型：
--
作者：
David K. Orren;Aziz Sancar
UvrA、UvrB和UvrC蛋白在ATP依赖性反应中共同催化受损DNA链的双切割。我们以前报道过（Orren，D. K.，和Sancar，A.等人（1989）Proc. Acad. Sci. U.S.A.86，5237-5241），UvrA将UvrB递送至DNA中的受损位点;在将UvrC添加至这些UvrB.DNA复合物后，DNA被切割。在本研究中，我们进一步表征了UvrB到DNA的传递和随后的切割过程，重点是ATP在这些反应中的作用。依赖于UvrA的UvrB向受损DNA的传递相对缓慢（kon约为6 × 10（4）M-1 s-1），需要ATP水解（Km = 120 μ M）。虽然ATP增强了UvrB.DNA复合物的稳定性（koff = 8.5 × 10（-5）s-1），但分离的UvrB.DNA复合物不含任何共价连接或稳定结合的核苷酸。然而，ATP结合是需要的UvrB结合的DNA的UvrC依赖性双切口。有趣的是，腺苷5 '-（3-O-硫代）三磷酸可以在这一步取代ATP。切割期间ATP的Km为2 μ M，但在切割反应期间ATP不会以可检测的水平水解。由UvrB-UvrC产生的切口位于加合物的两侧，并导致受损核苷酸的切除。
The UvrA, UvrB, and UvrC proteins collectively catalyze the dual incision of a damaged DNA strand in an ATP-dependent reaction. We previously reported (Orren, D. K., and Sancar, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5237-5241) that UvrA delivers UvrB to damaged sites in DNA; upon addition of UvrC to these UvrB.DNA complexes, the DNA is incised. In the present study, we have further characterized both the delivery of UvrB to DNA and the subsequent incision process, with emphasis on the role of ATP in these reactions. The UvrA-dependent delivery of UvrB onto damaged DNA is relatively slow (kon approximately 6 x 10(4) M-1 s-1) and requires ATP hydrolysis (Km = 120 microM). Although ATP enhances the stability of UvrB.DNA complexes (koff = 8.5 x 10(-5) s-1), the isolated UvrB.DNA complexes do not contain any covalently attached or stably bound nucleotide. However, ATP binding is required for the UvrC-dependent dual incision of DNA bound by UvrB. Interestingly, adenosine 5'-(3-O-thio)triphosphate can substitute for ATP at this step. The Km for ATP during incision is 2 microM, but ATP is not hydrolyzed at a detectable level during the incision reaction. The incisions made by UvrB-UvrC are on both sides of the adduct and result in the excision of the damaged nucleotide.