Independent mechanisms of stimulation of polynucleotide kinase/phosphatase by phosphorylated and non-phosphorylated XRCC1

Abstract

XRCC1 plays a central role in mammalian single-strand break repair. Although it has no enzymatic activity of its own, it stimulates the activities of polynucleotide kinase/phosphatase (PNKP), and this function is enhanced by protein kinase CK2 mediated phosphorylation of XRCC1. We have previously shown that non-phosphorylated XRCC1 stimulates the kinase activity of PNKP by increasing the turnover of PNKP. Here we extend our analysis of the XRCC1-PNKP interaction taking into account the phosphorylation of XRCC1. We demonstrate that phosphorylated and non-phosphorylated XRCC1 interact with different regions of PNKP. Phosphorylated XRCC1 binds with high affinity (K(d) = 3.5 nM and 1 : 1 stoichiometry) to the forkhead associated (FHA) domain, while non-phosphorylated XRCC1 binds to the catalytic domain of PNKP with lower affinity (K(d) = 43.0 nM and 1 : 1 stoichiometry). Under conditions of limited enzyme concentration both forms of XRCC1 enhance the activities of PNKP, but the effect is more pronounced with phosphorylated XRCC1, particularly for the kinase activity of PNKP. The stimulatory effect of phosphorylated XRCC1 on PNKP can be totally inhibited by the presence of excess FHA domain polypeptide, but non-phosphorylated XRCC1 is not susceptible to competition by the FHA domain. Thus, XRCC1 can stimulate PNKP by two independent mechanisms.

Figure 1.

In vitro phosphorylation of XRCC1. (A) ³²P-labeling demonstrating the phosphorylation of XRCC1 by CK2. (B) Linear MALDI-TOFMS analysis demonstrating the molecular weight increase of XRCC1 after phosphorylation. The upper trace shows the spectrum for pXRCC1 and the lower trace is for the npXRCC1. The peak with an m/z value of 66 477 shows the BSA calibration standard. (C) Linear MALDI-TOFMS analysis of the trypsin digestion products indicating multiple phosphorylation within peptide segments P1 (459–494) and P2 (503–546). Upper trace pXRCC1, lower trace npXRCC1. (D) Reflective MALDI-TOFMS analysis of the trypsin digest indicating multiple phosphorylation of peptide segment P3 (401–427). Upper trace pXRCC1, lower trace npXRCC1.

Figure 2.

The FHA domain of PNKP can interact with XRCC1 phosphorylated in vitro by CK2 and in A549 cell lines. (A) Phosphorylated XRCC1 (pXRCC1) and non-phosphorylated XRCC1 (npXRCC1) was incubated with the FHA domain in different molar ratios and then immunoprecipitated. The figure shows the results of western blots to determine if the FHA domain co-precipitated with the XRCC1 proteins. (B) Co-immunoprecipitation of the FHA domain after incubation with A549 cell lysate and immunoprecipitation with anti-XRCC1 antibodies, indicating the presence of phosphorylated XRC1 in A549 cells.

Figure 3.

Interaction of XRCC1 with different domains of PNKP. (A) Fluorescence titration of the AC labeled FHA domain versus pXRCC1. FHA-AC (0.065 µM) was excited at 380 nm and the relative fluorescence (Rel.Fluor) intensities were monitored at 480 nm (see inset). The fraction bound versus pXRCC1 concentration is plotted. (B) Sample plot of fluorescence data from titration with pXRCC1. F₀, F and F_∞ are the relative fluorescence intensities at 480 nm of FHA-AC alone, FHA-AC in the presence of a given concentration of pXRCC1, and FHA-AC saturated with pXRCC1, respectively. The plot is according to Chipman et al. (32). (C) Fluorescence titration of the AC labeled PNKP CT-domain versus npXRCC1. CT-AC (0.3 μM) was excited at 380 nm and the relative fluorescence (Rel. Fluor) intensities were monitored at 480 nm (see inset). The fraction bound versus npXRCC1 concentration is plotted. (D) Analysis of the fluorescence titration data of CT-AC with npXRCC1 as described for FHA-AC in panel B.

Figure 4.

Protein analysis by circular dichroism showing interaction of the catalytic domain of PNKP with non-phosphorylated XRCC1. Far-UV CD spectra of npXRCC1 (A) and PNKP-CT (B) in 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl₂. (C) Experimentally observed (filled square) and theoretical (filled triangle) CD spectra of the PNKP-CT:XRCC1 complex. For the theoretical spectrum the proteins are assumed to be non-interacting.

Figure 5.

Binding of pXRCC1 to DNA. Fluorescence titration of pXRCC1 with a 45mer single-stranded oligonucleotide. pXRCC1 (0.4 μM) in 50 mM Tris–HCl (pH 7.5), 100 mM NaCl and 5 mM MgCl₂ was excited at 295 nm, and the intrinsic fluorescence intensity was monitored as a function of DNA concentration at 340 nm (see inset). The fraction bound (i.e. relative fluorescence quenching) versus ligand concentration is plotted.

Figure 6.

Simultaneous monitoring of kinase and phosphatase activities of PNKP using the fluorescence-based assay of Dobson and Allinson (29). A nicked DNA substrate (80 nM) bearing a 3′-phosphate (P) and 5′-hydroxyl group (OH) was incubated with 0.1 mM ATP and 20 nM PNKP. The products of the reaction, i.e. FAM-labeled 18mer (3′-phosphatase product) and TAMRA-labeled p21mer (5′-kinase product) were monitored by gel electrophoresis (A) and quantified (B) over time.

Figure 7.

Comparison of PNKP activity with different substrates. Substrates (80 nM) bearing a nick (A), recessed 5′-terminus (B) and 1-nucleotide gap (C) were incubated with 4 nM PNKP and 0.1 mM ATP. (D) Plot of percentage of product accumulated over time. (open circle) 3′-dephosphorylation product from the nick substrate; (open square) 3′-dephosphorylation product from the gap substrate; (filled square) 5′-phosphorylation product from the gap substrate; (filled triangle) 5′-phosphorylation product from the recessed substrate. The data points represent the mean of three individual determinations and the error bars show the standard deviation from the mean.

Figure 8.

Stimulation of PNKP end-processing activities by pXRCC1 and npXRCC1. (A) The effect of pXRCC1 and npXRCC1 on PNKP phosphatase activity. The fluorescently labeled nick substrate (80 nM) was incubated with 4 nM PNKP and 0.1 mM ATP for 20 min and then divided into four aliquots. Three of the aliquots were supplemented with 20 nM pXRCC1, npXRCC1 or BSA and the fourth received buffer only. The reactions were followed for a further 20 min. (B) The effect of both pXRCC1 and npXRCC1 on the kinase activity of PNKP acting on the 5′OH group of a single-stranded 24-mer, using the same conditions as described above. The data were obtained from three individual determinations and the error bars represent the standard deviation from the mean.

Figure 9.

Competitive binding of the FHA domain to pXRCC1 abrogates the stimulation of PNKP kinase and phosphatase by pXRCC1. PNKP (8 nM) was incubated with either npXRCC1 or pXRCC1 (40 nM) in the absence or presence of FHA domain polypeptide (200 nM) at 4°C for 15 min and then allowed to react with the nick substrate (80 nM) at 37°C for 20 min and the kinase (dark shade) and phosphatase (light shade) activities were measured. The data points represent the mean of three individual determinations and the error bars show the standard deviation from the mean.