Mishra Subhra, Mazumdar Pooja Anjali, Dey Joykrishna, Das Amit Kumar
Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India.
Biochem Biophys Res Commun. 2003 Dec 19;312(3):615-22. doi: 10.1016/j.bbrc.2003.10.164.
The protein RecA is involved in homologous recombination, DNA repair and also catalyzes DNA strand exchange. RecX gene is downstream of recA and the gene product RecX is supposed to be important for RecA regulation. Recombinant RecX is purified to homogeneity, and circular dichroism (CD) and FTIR spectroscopy show the protein to exist mostly in helical conformation. The fluorescence emission maxima of the native and the denatured protein and the steady-state fluorescence quenching studies with acrylamide indicate the presence of tryptophan residues partially exposed to the bulk solvent. Denaturation studies with urea and guanidine hydrochloride by use of spectroscopic methods, fluorescence, and CD also confirm the instability of the protein and unfolding occurs following a two-state model. Mass spectrometry and gel permeation chromatography suggest the monomeric form of the protein. Molecular modeling of RecX represents the molecule as extended and helical bundle in conformity with the spectroscopic results. To understand the mechanism of RecX in the regulation of RecA the structural model of RecA-RecX has been discussed. In this proposed model, entry of RecX into hexameric RecA filament prevents binding of ssDNA and also inhibits ATPase activity.
蛋白质RecA参与同源重组、DNA修复，还催化DNA链交换。RecX基因位于recA下游，其基因产物RecX被认为对RecA的调控很重要。重组RecX被纯化至同质，圆二色性（CD）和傅里叶变换红外光谱（FTIR）表明该蛋白质主要以螺旋构象存在。天然和变性蛋白质的荧光发射最大值以及用丙烯酰胺进行的稳态荧光猝灭研究表明存在部分暴露于溶剂中的色氨酸残基。通过光谱方法、荧光和CD对尿素和盐酸胍进行的变性研究也证实了该蛋白质的不稳定性，且其展开遵循二态模型。质谱和凝胶渗透色谱表明该蛋白质为单体形式。RecX的分子模型显示该分子呈伸展的螺旋束状，与光谱结果一致。为了解RecX在RecA调控中的机制，已对RecA-RecX的结构模型进行了讨论。在这个提出的模型中，RecX进入六聚体RecA细丝会阻止单链DNA的结合，还会抑制ATP酶活性。
