Purification and preliminary characterization of the Escherichia coli K-12 recF protein.
The recF gene of Escherichia coli is known to encode an Mr-40,000 protein that is involved in DNA recombinationa nd postreplication DNA repair. To characterize the role of the recF gene product in these processes, the recF gene was cloned downstream of a tac promoter to facilitate overproduction of the recF gene product. The RecF protein was overproduced and purified to apparent homogeneity. N-terminal protein sequence analysis demonstrated that the purified protein had the sequence that was predicted from the DNA sequence of the recF gene, except that the predicted N-terminal Met was not present. The RecF protein bound to single-stranded oligonucleotides in filter binding and gel filtration assays. Maximal binding required 2 to 3 min of incubation at 37 degrees C; the binding reaction had a pH optimum of 7.0, did not require divalent cations, and was inhibited by NaCl concentrations of greater than 250 mM. The Kd of RecF protein binding to a 59-base single-stranded oligonucleotide was on the order of 1.3 X 10(-7) M, and the reaction did not show cooperativity. Experiments measuring the binding to various DNA substrates and competition binding experiments with different DNA molecules demonstrated that RecF protein binds preferentially to single-stranded, linear DNA molecules.
Griffin
T J
TJ
Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Kolodner
R D
RD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010957:Plasmids; D015183:Restriction Mapping; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015345:Oligonucleotide Probes; C489340:recF protein, E coli; C066218:recF protein, Bacteria