Sugitani Norie, Voehler Markus W, Roh Michelle S, Topolska-Woś Agnieszka M, Chazin Walter J
From the Departments of Chemistry and.
the Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-7917.
J Biol Chem. 2017 Oct 13;292(41):16847-16857. doi: 10.1074/jbc.M117.800078. Epub 2017 Aug 31.
Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.
着色性干皮病（XP）互补组A（XPA）是多蛋白核苷酸切除修复（NER）机制中一种重要的支架蛋白。XPA与DNA的相互作用是该蛋白的核心功能；DNA结合结构域（DBD）中的一些突变与XP疾病相关。尽管已经报道了人类XPA中央球状结构域的结构以及DNA底物结合的数据，但XPA DNA结合活性的结构基础仍然未知。酵母XPA（Rad14）中央球状结构域与含损伤DNA双链体的X射线晶体结构提供了有价值的见解，但本研究中使用的DNA底物与XPA在NER机制中发挥功能时的底物不对应。为了更好地理解人类XPA在NER中的DNA结合活性，我们使用核磁共振（NMR）研究了其DBD与一系列DNA底物的相互作用。我们发现XPA通过一个共同的表面结合不同的单链/双链连接DNA底物。将我们基于NMR的结合残基图谱与先前报道的Rad14-DNA晶体结构进行比较，揭示了XPA和Rad14在底物结合方面的异同。这包括DNA与从球状核心向C端延伸的残基接触的直接证据，而Rad14构建体中缺少这些残基。此外，XPA中对应于Rad14中Phe-262的残基发生突变，此前报道该残基对DNA结合至关重要，但对XPA的DNA结合活性只有中等程度的影响。我们研究了DBD中几种与疾病相关的突变的DNA结合特性。这些结果表明，对于表现出改变的DNA结合特性的XPA突变体，DNA结合亲和力降低的程度与XP患者症状的严重程度之间存在相关性。
