Interactions of human nucleotide excision repair protein XPA with DNA and RPA70ΔC327:: Chemical shift mapping and 15N NMR relaxation studies
Interactions of human nucleotide excision repair protein XPA with DNA and RPA70ΔC327:: Chemical shift mapping and 15N NMR relaxation studies
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DOI：
10.1021/bi991755p
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发表时间：
1999-11-16
期刊：
影响因子：
2.9
通讯作者：
Kennedy, MA
中科院分区：
文献类型：
--
作者：
Buchko, GW;Daughdrill, GW;Kennedy, MA
人类XPA是多酶核苷酸切除修复（NER）途径的重要组成部分。最近确定了XPA最小DNA结合域（XPA- mbd: M98-F219）的溶液结构[Buchko et al.（1998）核酸研究，26,2779-2788,Ikegami et al. (1998) Nat. Struct。Biol. 5,701 -706]，由紧凑的锌结合核心和由连接子序列连接的富含环的c端亚域组成。在这里，XPA-MBD的溶液结构使用了一种全新的基于钴取代XPA-MBD测量的假接触位移的约束进一步完善，使用这种结构，XPA-MBD与DNA和复制蛋白a的最大亚基片段（RPA70 Delta C327: M1-Y326）相互作用的表面使用化学位移作图确定。XPA- mbd中的DNA结合高度定位于有或没有病变的DNA[二氢胸腺嘧啶（dhT）或6-4胸腺嘧啶-胞苷（64TC）]的富含环的亚结构域，或者DNA以单链或双链形式结合，这表明病变本身的特征并不是XPA结合DNA的驱动力。RPA70 Delta C327在锌结合区和富环子结构域均有接触，在富环子结构域DNA与RPA70 Delta C327的结合区有部分重叠，提示XPB与RP870 Delta C327可能存在协同结合模式。为了补充化学位移映射数据，利用N-15核磁共振松弛数据研究了游离XPA-MBD和XPA-MBD与含有dhT或64TC病变的DNA低聚物结合的主链动力学。对XPA-MBD复合物与DNA的动态分析表明，S-2的局部升高和降低以及全局相关时间的增加。XPA-MBD中S-2重叠区增加最多的区域在结合DNA后化学位移变化最大，表明富环亚结构域在结合DNA后变得更加刚性。有趣的是，在DNA结合后，锌结合核心中一些残基的S-2减少，表明可能在结合DNA上发生了协同结构重排。
Human XPA is an essential component in the multienzyme nucleotide excision repair (NER) pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219) was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779-2788, Ikegami et al, (1998) Nat. Struct. Biol. 5, 701-706] and shown to consist of a compact zinc-binding core and a loop-rich C-terminal subdomain connected by a linker sequence. Here, the solution structure of XPA-MBD was further refined using an entirely new class of restraints based on pseudocontact shifts measured in cobalt-substituted XPA-MBD, Using this structure, the surface of XPA-MBD which interacts with DNA and a fragment of the largest subunit of replication protein A (RPA70 Delta C327: M1-Y326) was determined using chemical shift mapping. DNA binding in XPA-MBD was highly localized in the loop-rich subdomain for DNA with or without a lesion [dihydrothymidine (dhT) or 6-4-thymidine-cytidine (64TC)], or with DNA in single- or- double-stranded form, indicating that the character of the lesion itself is not the driving force for XPA binding DNA. RPA70 Delta C327 was found to contact regions in both the zinc-binding and loop-rich subdomains, Some overlap of the DNA and RPA70 Delta C327 binding regions was observed in the loop-rich subdomain, indicating a possible cooperative DNA-binding mode between XPB and RP870 Delta C327. To complement the chemical shift mapping data, the backbone dynamics of free XPA-MBD and XPA-MBD bound to DNA oligomers containing dhT or 64TC lesions were investigated using N-15 NMR relaxation data. The dynamic analyses for the XPA-MBD complexes with DNA revealed localized increases and decreases in S-2 and an increase in the global correlation time. Regions of XPA-MBD with the largest increases in S-2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicating that the loop-rich subdomain becomes more rigid upon binding DNA, Interestingly, S-2 decreased for some residues in the zinc-binding core upon DNA association, indicating a possible concerted structural rearrangement on binding DNA,