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

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Oxford University Press

RESUMO

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 α-helix-3).

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