Chemical shift changes provide evidence for overlapping single-stranded DNA- and XPA-binding sites on the 70 kDa subunit of human replication protein A.
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.
Daughdrill
Gary W
GW
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, PO Box 443052, Life Science South Room 142, Moscow, ID 83844-3052, USA. gdaugh@uidaho.edu
Buchko
Garry W
GW
Botuyan
Maria V
MV
Arrowsmith
Cheryl
C
Wold
Marc S
MS
Kennedy
Michael A
MA
Lowry
David F
DF
D001665:Binding Sites / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A; D051760:Xeroderma Pigmentosum Group A Protein
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; C497167:RPA1 protein, human; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein