Nucleic Acids Research, 2001, Vol. 29, No. 12 2635-2643
© 2001 Oxford University Press
DNA–XPA interactions: a 31P NMR and molecular modeling study of dCCAATAACC association with the minimal DNA-binding domain (M98–F219) of the nucleotide excision repair protein XPA
1Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA, 2Departments of Biochemistry and Radiobiology, Duke University Medical Center, Durham, NC 27710, USA and 3Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
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 31P 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 31P 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 15N (XPA-MBD) and 31P (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 
-helix-3).
* To whom correspondence should be addressed. Tel: +1 509 372 2168; Fax: +1 509 376 2303; Email: ma_kennedy{at}pnl.gov
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