Luisi-DeLuca C, Kolodner R
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, PA 15261.
J Mol Biol. 1994 Feb 11;236(1):124-38. doi: 10.1006/jmbi.1994.1123.
The recO gene product is required for RecF pathway-mediated recombination and the repair of DNA damage after UV irradiation or mitomycin C exposure in Escherichia coli. In this study, the E. coli recO gene product was overexpressed and purified to at least 99% homogeneity. N-Terminal protein sequence analysis of the overexpressed 31 kDa polypeptide confirmed that this polypeptide was encoded by the recO gene. The N-terminal protein sequence of RecO also confirmed that the first 12 amino acids of functional RecO protein are encoded within the upstream era gene. The purified protein chromatographs with the same Stokes radius (25 A) as a globular protein having a molecular mass of 28 kDa, indicating that RecO is a monomer in solution. The purified RecO protein binds to both single-stranded and double-stranded DNA, and promotes renaturation of complementary single-stranded DNA molecules in the absence of any high energy cofactor. The rate constant for this reaction is independent of the concentration of DNA, suggesting that the reaction follows first-order reaction kinetics. In addition, this reaction is inhibited by 160 mM NaCl, requires Mg2+, and is not stimulated by ATP. These biochemical characteristics support a role for RecO protein in an early phase of homologous recombination.
RecO基因产物是大肠杆菌中RecF途径介导的重组以及紫外线照射或丝裂霉素C处理后DNA损伤修复所必需的。在本研究中，大肠杆菌recO基因产物被过量表达并纯化至至少99%的纯度。对过量表达的31 kDa多肽进行N端蛋白质序列分析，证实该多肽由recO基因编码。RecO的N端蛋白质序列还证实，功能性RecO蛋白的前12个氨基酸是由上游era基因编码的。纯化后的蛋白质与分子量为28 kDa的球状蛋白具有相同的斯托克斯半径（25 Å），表明RecO在溶液中是单体。纯化后的RecO蛋白能与单链和双链DNA结合，并在没有任何高能辅因子的情况下促进互补单链DNA分子的复性。该反应的速率常数与DNA浓度无关，表明该反应遵循一级反应动力学。此外，该反应受到160 mM NaCl的抑制，需要Mg2+，且不受ATP的刺激。这些生化特性支持RecO蛋白在同源重组早期阶段发挥作用。
