The human checkpoint sensor and alternative DNA clamp Rad9-Rad1-Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery

Abstract

The human checkpoint sensor and alternative clamp Rad9-Rad1-Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9-Rad1-Hus1 with MutY DNA glycosylase, DNA polymerase beta and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9-Rad1-Hus1 complex, thus enhancing the quality control of DNA.

Figure 1. The human 9-1-1 complex interacts with human DNA ligase I

Pull-down of the 9-1-1 complex and DNA ligase I. (A) His pull-down experiments were performed in the presence of either His-tagged DNA ligase I (5 μg) or nickel beads alone, and purified 9-1-1 complex (3.6 μg). (B) The control His pull-down experiments were performed in the presence of either His-tagged DNA ligase I (5 μg) or nickel beads alone, and purified GST (3.6 μg). The presence of co-precipitated proteins was determined by SDS/PAGE followed by Western blot (WB) analysis. lig I, DNA ligase I.

Figure 2. The human 9-1-1 complex can stimulate human DNA ligase I

DNA ligase activity was determined as described in the Materials and methods section. (A) Substrate used in DNA ligase assays. (B) SDS/PAGE, followed by Coomassie Blue staining of the purified His-9-1-1 complex used in the assays. MW, molecular-mass values. (C) Effect of the 9-1-1 complex on DNA ligase I activity. The assays were performed in the presence of 30 fmol of recombinant human DNA ligase I. Lane 1, 46-mer marker; lane 2, no enzyme control, 25-mer marker; lane 3, DNA ligase I alone; lanes 4–7, 30, 90, 270 or 810 fmol respectively of the purified recombinant human 9-1-1 complex was added; lane 8, no enzyme control; lane 9, DNA ligase I alone; lanes 10–13, 30, 90, 270 or 810 fmol respectively of myoglobin was added. (D) Quantification of the effect of the 9-1-1 complex (lanes 3–7 in C) and of myoglobin (lanes 9–13 in C) on DNA ligase I activity. The data represent the means for three experiments.

Figure 3. Human PCNA can slightly stimulate human DNA ligase I, but cannot prevent the stimulation of DNA ligase I by the human 9-1-1 complex

DNA ligase activity was determined as described in the Materials and methods section. (A) Effect of the 9-1-1 complex and PCNA on the activity of human DNA ligase I. The assays were performed in the presence of 30 fmol of recombinant human DNA ligase I. Lane 1, 46-mer marker; lane 2, no enzyme control, 25-mer marker; lane 3, DNA ligase I alone; lanes 4–7, 30, 90, 270 or 810 fmol respectively of the purified recombinant human 9-1-1 complex was added; lane 8, 2430 fmol of 9-1-1 complex was added in the absence of DNA ligase I, no ligase control; lane 9, DNA ligase I alone; lanes 10–13, 30, 90, 270 or 810 fmol of the purified recombinant human PCNA was added. (B) Effect of PCNA on the stimulation of DNA ligase I by the 9-1-1 complex. The assays were performed in the presence of 30 fmol of recombinant human DNA ligase I. Lane 1, no enzyme control, 25-mer marker; lane 2, DNA ligase I alone; lanes 3–6, DNA ligase in the presence of 270 fmol of PCNA and 0, 90, 270 or 810 fmol respectively of the 9-1-1 complex; lane 7, no enzyme control, 25-mer marker; lane 8, DNA ligase I alone; lanes 9–12, DNA ligase I and 270 fmol of 9-1-1 complex in the presence of 90, 270 or 810 fmol respectively of PCNA. (C) 1, Quantification of the effect of the 9-1-1 complex alone (lanes 3–7 in A); 2, quantification of the effect of PCNA alone (lanes 9–13 in A); 3, quantification of the stimulation by the 9-1-1 complex in the presence of PCNA (lanes 3–6 in B); 4, quantification of the PCNA effect in the presence of the 9-1-1 complex (lanes 9–12 in B). The data presented in (C) are the means for three experiments.