THE (A)BC EXCINUCLEASE OF ESCHERICHIA-COLI HAS ONLY THE UVRB AND UVRC SUBUNITS IN THE INCISION COMPLEX
THE (A)BC EXCINUCLEASE OF ESCHERICHIA-COLI HAS ONLY THE UVRB AND UVRC SUBUNITS IN THE INCISION COMPLEX
复制标题
DOI：
10.1073/pnas.86.14.5237
复制
发表时间：
1989-07-01
影响因子：
11.1
通讯作者：
SANCAR, A
中科院分区：
文献类型：
--
作者：
ORREN, DK;SANCAR, A
uvrA、uvrB和uvrC基因控制大肠杆菌中的切除修复。这三个基因中任何一个突变的细胞都不能通过核苷酸切除来修复DNA。当将纯化的基因产物<$bUvrA、UvrB和UvrC蛋白<$bD混合在一起时，形成切除核酸酶，该切除核酸酶在ATP依赖性反应中在受损核苷酸的两侧上切割;已经推测切除核酸酶是含有所有三种Uvr蛋白的ABC复合物。为了确定酶中亚基的化学计量，我们用有或没有DNA底物的亚基混合物进行了流体动力学研究。我们发现，没有DNA的UvrA亚基是一个二聚体，当UvrB蛋白也存在，（UvrA）2（UvrB）1复合物的形式。在没有DNA的情况下，UvrA或UvrB亚基或（UvrA）2（UvrB）1复合物与UvrC亚基之间没有可检测到的相互作用。出乎意料的是，用UV照射的DNA，分离的DNA-蛋白质复合物中的UvrA/UvrB比率是可变的，并且该比率随着反应混合物中UvrA浓度的降低而变得无限低。在饱和UvrB蛋白的条件下，在需要催化量的UvrA亚基的反应中，每个受损位点大约有一个UvrB分子与DNA结合。将UvrC蛋白添加到纯化的UvrB-DNA复合物中导致DNA的快速切割，推测是由仅含有UvrB和UvrC亚基的切除核酸酶催化的。
The uvrA, uvrB, and uvrC genes control excision repair in Escherichia coli. Cells with mutations in any of these three genes cannot repair DNA by nucleotide excision. When the purified gene products.sbd.the UvrA, UvrB, and UvrC proteins.sbd.are mixed together, an excision nuclease is formed that incises on both sides of the damaged nucleotide in an ATP-dependent reaction; it has been presumed that the excision nuclease was an ABC complex containing all three Uvr proteins. To determine the stoichiometry of the subunits in the enzyme, we conducted hydrodynamic studies with mixtures of the subunits with or without DNA substrate. We found that without DNA the UvrA subunit is a dimer and that when UvrB protein is also present, a (UvrA)2(UvrB)1 complex forms. Without DNA no detectable interaction of either the UvrA or UvrB subunits or the (UvrA)2(UvrB)1 complex with the UvrC subunit occurs. Unexpectedly, with UV-irradiated DNA, the UvrA/UvrB ratio in isolated DNA-protein complexes is variable, and the ratio becomes infinitesimally low as the UvrA concentration in the reaction mixture decreases. Under conditions of saturating UvrB protein approximately one UvrB molecule binds to DNA per damaged site in a reaction that requires catalytic amounts of UvrA subunit. Addition of UvrC protein to purified UvrB-DNA complexes results in rapid incision of the DNA, presumably catalyzed by an excision nuclease containing only UvrB and UvrC subunits.