Purification and Preliminary Characterization of the Escherichia Coli K-12 RecF Protein

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 1990 Nov 1

PMID 2228960

Citations 46

Authors

T J Griffin 4th

R D Kolodner

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Generation and Repair of Postreplication Gaps in Escherichia coli.

Cox M, Goodman M, Keck J, van Oijen A, Lovett S, Robinson A Microbiol Mol Biol Rev. 2023; 87(2):e0007822.

PMID: 37212693 PMC: 10304936. DOI: 10.1128/mmbr.00078-22.

RecF protein targeting to post-replication (daughter strand) gaps II: RecF interaction with replisomes.

Henry C, Kaur G, Cherry M, Henrikus S, Bonde N, Sharma N Nucleic Acids Res. 2023; 51(11):5714-5742.

PMID: 37125644 PMC: 10287930. DOI: 10.1093/nar/gkad310.

RecF protein targeting to postreplication (daughter strand) gaps I: DNA binding by RecF and RecFR.

Henry C, Mbele N, Cox M Nucleic Acids Res. 2023; 51(11):5699-5713.

PMID: 37125642 PMC: 10287957. DOI: 10.1093/nar/gkad311.

Mechanism of RecF-RecO-RecR cooperation in bacterial homologous recombination.

Nirwal S, Czarnocki-Cieciura M, Chaudhary A, Zajko W, Skowronek K, Chamera S Nat Struct Mol Biol. 2023; 30(5):650-660.

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Elucidating Recombination Mediator Function Using Biophysical Tools.

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