DNA-XPA interactions: a (31)P NMR and molecular modeling study of dCCAATAACC association with the minimal DNA-binding domain (M98-F219) of the nucleotide excision repair protein XPA.
Recent NMR-based, chemical shift mapping experiments with the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) suggest that a basic cleft located in the loop-rich subdomain plays a role in DNA-binding. Here, XPA-DNA interactions are further characterized by NMR spectroscopy from the vantage point of the DNA using a single-stranded DNA nonamer, dCCAATAACC (d9). Up to 2.5 molar equivalents of XPA-MBD was titrated into a solution of d9. A subset of (31)P resonances of d9 were observed to broaden and/or shift providing direct evidence that XPA-MBD binds d9 by a mechanism that perturbs the phosphodiester backbone of d9. The interior five residues of d9 broadened and/or shifted before (31)P resonances of phosphate groups at the termini, suggesting that when d9 is bound to XPA-MBD the internal residues assume a correlation time that is characteristic of the molecular weight of the complex while the residues at the termini undergo a fraying motion away from the surface of the protein on a timescale such that the line widths are more characteristic of the molecular weight of ssDNA. A molecular model of the XPA-MBD complex with d9 was calculated based on the (15)N (XPA-MBD) and (31)P (d9) chemical shift mapping studies and on the assumption that electrostatic interactions drive the complex formation. The model shows that a nine residue DNA oligomer fully covers the DNA-binding surface of XPA and that there may be an energetic advantage to binding DNA in the 3'-->5' direction rather than in the 5'-->3' direction (relative to XPA-MBD alpha-helix-3).
Buchko
G W
GW
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Tung
C S
CS
McAteer
K
K
Isern
N G
NG
Spicer
L D
LD
Kennedy
M A
MA
D001483:Base Sequence; D001665:Binding Sites; D004260:DNA Repair / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D055672:Static Electricity; D017186:Titrimetry; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D051760:Xeroderma Pigmentosum Group A Protein
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011522:Protons; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein