Aberrant mobility phenomena of the DNA repair protein XPA
Aberrant mobility phenomena of the DNA repair protein XPA
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DOI：
10.1110/ps.40101
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发表时间：
2001-07-01
期刊：
影响因子：
8
通讯作者：
Ackerman, EJ
中科院分区：
文献类型：
--
作者：
Iakoucheva, LM;Kimzey, AL;Ackerman, EJ
DNA修复蛋白XPA识别各种大小的病变，并在核苷酸切除修复过程中与其他几种蛋白质相互作用。我们最近利用时间分辨胰酶蛋白分解和电喷雾电离界面与傅立叶变换离子回旋共振(ESI-FTICR)质谱仪(MS)相结合的实验方法，确定了非洲爪哇XPA(XXPA)全长蛋白质的内在有序和无序区域。MS数据与xXPA不含翻译后修饰的解释一致。在这里，我们表征了计算的xXPA分子量(31 KDa)与其在SDS-PAGE(多条带，从类似40-45 kDa)和凝胶过滤层析(类似于92 kDa)上的表观分子量之间的差异，以及DNA结合对其异常迁移率的影响。碘乙酰胺处理后的xXPA在SDS-PAGE前产生一条42 kDa的单一条带，表明Cys的共价修饰不能纠正异常的迁移率。用Ellman试剂测定xXPA中的巯基含量表明，活性蛋白中的9种半胱氨酸都减少了。出人意料的是，xXPA中分子内戊二醛交联引起的结构约束产生了类似于32 kDa的单体，与其计算的分子量更接近。为了研究与DNA的结合是否改变了xXPA的异常迁移，我们使用了凝胶过滤层析。我们首次提纯了xXPA和DNA+/-顺铂+/-错配的稳定络合物。与未受损的DNA F错配相比，xXPA对顺铂DNA+/-错配的亲和力至少高出10倍。在所有情况下，DNA结合都不能纠正xXPA的异常迁移。为了验证富含Glu区域(EEAEE)和/或无序的N-末端和C-末端结构域是导致xXPA异常迁移率的原因这一预测，用反相高效液相色谱法分离并通过ESI-FTICR MS精确测定的部分蛋白水解区的分子量与其在SDS-PAGE上的迁移量相关联，每个在此大小范围内分析的部分胰酶片段显示的分子量比预期大10%-50%，因此，xXPA的无序结构域和富含Glu区域都是导致异常迁移率的主要原因。
The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time-resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post-translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS-PAGE (multiple bands from similar to 40-45 kDa) and gel filtration chromatography (similar to 92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a similar to 32-kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA +/- cisplatin +/- mismatches. xXPA showed at least 10-fold higher affinity for cisplatin DNA +/- mismatches compared to undamaged DNA F mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu-rich region (EEEEAEE) and/or disordered N- and C-terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from similar to5 to 25 kDa separated by reverse-phase HPLC and precisely determined by ESI-FTICR MS were correlated with their migration on SDS-PAGE, Every partial tryptic fragment analyzed within this size range exhibited 10%-50% larger molecular weights than expected, Thus, both the disordered domains and the Glu-rich region in xXPA are primarily responsible for the aberrant mobility phenomena.