Purification and characterization of the Escherichia coli RecO protein. Renaturation of complementary single-stranded DNA molecules catalyzed by the RecO protein.
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.
Luisi-DeLuca
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C
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, PA 15261.
Kolodner
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R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D017886:Durapatite; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D009695:Nucleic Acid Renaturation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; C081006:RecO protein, E coli; D017886:Durapatite