Matsunaga T, Mu D, Park C H, Reardon J T, Sancar A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599, USA.
J Biol Chem. 1995 Sep 1;270(35):20862-9. doi: 10.1074/jbc.270.35.20862.
Human DNA repair excision nuclease removes DNA damage by incising on both sides of the lesion in a precise manner. The activity requires participation of 16-17 polypeptides. Of these, the XPF.ERCC1 complex and XPG were predicted to carry the nuclease active sites based on studies with the recombinant proteins and the yeast homologs of these proteins. Furthermore, recent work with model (undamaged) substrates have led to predictions of the roles of these proteins in incising 5' or 3' to the lesion. We have used damaged DNA substrates and antibodies to XPG and ERCC1 to test these predictions. Our results reveal that anti-XPG antibodies change the site of 3' incision and at high concentration inhibit the 3' incision without significantly affecting the 5' incision, indicating that XPG makes the 3' incision and further that under this condition 5' incision can occur without 3' incision. In contrast, anti-ERCC1 antibodies inhibit both the 3' and 5' incisions. Using a defined system for excision repair we also demonstrate that the 3' incision can occur without the 5' incision, leading us to conclude that under certain conditions the two incisions can occur independently.
人类DNA修复切除核酸酶通过精确切割损伤部位两侧的DNA来去除DNA损伤。该活性需要16 - 17种多肽的参与。其中，基于对重组蛋白及这些蛋白的酵母同源物的研究，预测XPF.ERCC1复合物和XPG携带核酸酶活性位点。此外，最近对模型（未损伤）底物的研究得出了这些蛋白在损伤部位5'或3'端切割中所起作用的预测。我们使用损伤的DNA底物以及针对XPG和ERCC1的抗体来检验这些预测。我们的结果显示，抗XPG抗体改变了3'端切割位点，并且在高浓度时抑制3'端切割而不显著影响5'端切割，这表明XPG进行3'端切割，进一步表明在此条件下5'端切割可以在没有3'端切割的情况下发生。相比之下，抗ERCC1抗体抑制3'端和5'端切割。使用一个明确的切除修复系统，我们还证明了3'端切割可以在没有5'端切割的情况下发生，从而使我们得出结论，在某些条件下这两种切割可以独立发生。
