PMC

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.

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.

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.