PURIFICATION AND CHARACTERIZATION OF THE ESCHERICHIA-COLI RECO PROTEIN - RENATURATION OF COMPLEMENTARY SINGLE-STRANDED-DNA MOLECULES CATALYZED BY THE RECO PROTEIN
PURIFICATION AND CHARACTERIZATION OF THE ESCHERICHIA-COLI RECO PROTEIN - RENATURATION OF COMPLEMENTARY SINGLE-STRANDED-DNA MOLECULES CATALYZED BY THE RECO PROTEIN
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DOI：
10.1006/jmbi.1994.1123
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发表时间：
1994-02-11
影响因子：
5.6
通讯作者：
KOLODNER, R
中科院分区：
文献类型：
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作者：
LUISIDELUCA, C;KOLODNER, R
在大肠杆菌中，RECF途径介导的重组和紫外线照射或丝裂霉素C暴露后DNA损伤的修复需要该基因产物。在这项研究中，你。ColirecOgene表达产物得到高效表达和纯化，同源性至少达到99%以上。对过表达的31 kDa多肽进行N-末端蛋白序列分析，证实该多肽是由该基因编码的。RECO的N-末端蛋白序列也证实了RECO蛋白的前12个氨基酸编码在上游基因内。纯化蛋白的斯托克斯半径(25？)与相对分子质量为28 kDa的球状蛋白相同，表明Reco在溶液中为单体。纯化的Reco蛋白可与单链和双链DNA结合，并在没有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的浓度无关，表明该反应符合一级反应动力学。此外，这个反应被160 mM的氯化钠抑制，需要镁离子，并且不被三磷酸腺苷刺激。这些生化特性支持RECO蛋白在同源重组的早期阶段发挥作用。
TherecOgene product is required for RecF pathway-mediated recombination and the repair of DNA damage after UV irradiation or mitomycin C exposure inEscherichia coli. In this study, theE. coli recOgene 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 therecOgene. The N-terminal protein sequence of RecO also confirmed that the first 12 amino acids of functional RecO protein are encoded within the upstreameragene. The purified protein chromatographs with the same Stokes radius (25 Å) 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.