Chemical shift changes provide evidence for overlapping single-stranded DNA- and XPA-binding sites on the 70 kDa subunit of human replication protein A

Abstract

Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1-326 (hRPA70(1-326)), and a fragment of the human XPA protein, residues 98-219 (XPA-MBD). Intensity changes were observed for amide resonances in the (1)H-(15)N correlation spectrum of uniformly (15)N-labeled hRPA70(1-326) after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA70(1-326) fragment. The hRPA70(1-326) residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA70(1-326) suggests that a competitive binding mechanism mediates the formation of the RPA-XPA complex. To determine whether a ternary complex could form between hRPA70(1-326), XPA-MBD and ssDNA, a (1)H-(15)N correlation spectrum was acquired for uniformly (15)N-labeled hRPA70(1-326) after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA70(1-326) in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA70(1-326) suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA70(1-326) may be modulated by ssDNA.

Figure 1

Amide region of the ¹H–¹⁵N HSQC spectra of hRPA70_1–326 on the left, and hRPA70_1–326 after the addition of XPA-MBD on the right. Arrows show the positions for assigned resonances that had a 2-fold or greater intensity reduction after the addition of XPA-MBD. New resonances that were not assignable are circled. Both spectra were collected at 25°C and at a ¹H resonance frequency of 600 MHz.

Figure 2

Plot of the hRPA70_1–326 intensity ratios versus residue number for binding to XPA-MBD. I_i refers to the resonance intensity before the addition of XPA-MBD and I_f refers to the resonance intensity after the addition of an equimolar amount of XPA-MBD.

Figure 3

Log plot of the hRPA70_1–326 intensity ratios versus residue number for binding to CoXPA-MBD. I_i refers to the resonance intensity before the addition of CoXPA-MBD and I_f refers to the resonance intensity after the addition of an equimolar amount of CoXPA-MBD.

Figure 4

Ribbon models for residues 183–291 of SSB1 from hRPA70 adapted from the coordinates in PDB accession file 1jmc (22). (a) SSB1 residues with the largest resonance intensity reductions observed in the ¹H–¹⁵N HSQC spectrum of hRPA70_1–326 when bound to XPA-MBD and CoXPA-MBD are colored magenta and green, respectively. (b) SSB1 residues with the largest chemical shift changes in the NMR analysis of three different hRPA70_1–326–ssDNA complexes are colored blue and SSB1 residues that are in contact with ssDNA in the crystal structure but were not assignable in the NMR analysis of the hRPA70_1–326–ssDNA complexes are colored red (22,31).

Figure 5

Diagram showing the amino acid sequence and secondary structure of SSB1 residues 197–288. SSB1 residues colored red under the linear sequence made direct contacts with ssDNA in the crystal structure (22). SSB1 residues colored blue under the linear sequence had the largest chemical shift changes when hRPA70_1–326 was bound to ssDNA (31). SSB1 residues colored magenta under the linear sequence had the largest resonance intensity reductions when hRPA70_1–326 was bound to XPA-MBD. SSB1 residues colored green under the linear sequence had the largest resonance intensity reductions when hRPA70_1–326 was bound to CoXPA-MBD.

Figure 6

Selected regions from the ¹H–¹⁵N HSQC spectra for hRPA70_1–326. The figure shows the spectrum of hRPA70_1–326 in the absence of XPA-MBD and d9 (top left), in the presence of an equimolar amount of d9 (top right), in the presence of an equimolar amount of XPA-MBD (bottom left), and in the presence of an equimolar amount of XPA-MBD and d9 (bottom right).