Human DNA repair excision nuclease. Analysis of the roles of the subunits involved in dual incisions by using anti-XPG and anti-ERCC1 antibodies

Abstract

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.