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
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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DOI：
10.1093/nar/29.12.2635
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发表时间：
2001-06-15
影响因子：
14.9
通讯作者：
Kennedy, MA
中科院分区：
文献类型：
--
作者：
Buchko, GW;Tung, CS;Kennedy, MA
最近基于核磁共振的XPA最小dna结合域（XPA- mbd: M98-F219）化学位移定位实验表明，位于富环亚域的基本裂缝在dna结合中起作用。在这里，XPA-DNA的相互作用通过核磁共振光谱进一步表征，从DNA的有利位置使用单链DNA nonamer， dCCAATAACC （d9）。高达2.5摩尔当量的XPA-MBD被滴定到d9溶液中。观察到d9的P-31共振子集变宽和/或移位，这直接证明XPA-MBD通过一种扰乱d9磷酸二酯主链的机制结合d9。d9的内部5个残基在末端磷酸基P-31共振之前加宽和/或移位，这表明当d9与XPA-MBD结合时，内部残基的相关时间是复合物分子量的特征，而末端残基在时间尺度上经历了远离蛋白质表面的磨损运动，使得线宽更具有ssDNA分子量的特征。基于N-15 （XPA-MBD）和P-31 （d9）的化学位移映射研究，并假设静电相互作用驱动配合物的形成，计算了XPA-MBD与d9配合物的分子模型。该模型表明，9个残基DNA寡聚物完全覆盖了XPA的DNA结合表面，与XPA- mbd α -helix-3相比，在3‘- >5’方向上结合DNA比在5‘- >3’方向上结合DNA具有能量优势。
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 P-31 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 P-31 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 N-15 (XPA-MBD) and P-31 (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).