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.     

Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena

In recb recC sbcB mutants genetic recombination is dependent upon the recF gene. recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background. Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background. In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages. In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101. These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.     

Two-component suppression of recF143 by recA441 in Escherichia coli K-12.

Sensitivity to UV irradiation conferred by recF143 was partially suppressed by recA441 (also known as tif-1). A temperature-conditional component depended on uvrA function and is thought to involve thermal induction of excision repair enzymes. In a uvrA6 mutant, a temperature-independent component of suppression was seen. This is thought to indicate that recA441 also caused temperature-independent changes in recA activity. Two hypotheses are offered to explain how recA441 produced both thermosensitive and thermoindependent effects.     

Genetic and physical mapping of recF in Escherichia coli K-12

Summary Two factor transductional crosses place recF at approximately 82 min on the E. coli chromosome; recF is highly cotransducible with dnaA and gyrB (cou). Transductional analysis with a series of tna specialized transducing phages carrying chromosomal DNA from the tnaA region place recF between dnaA and gyrB. This analysis also indicates that a gene lying in the same region and producing an easily detectable protein (estimated MW of 45 kD) is dnaN and not recF.     

recA (Srf) suppression of recF deficiency in the postreplication repair of UV-irradiated Escherichia coli K-12.

The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter-strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein.     

Escherichia coli K-12 mutants in which viability is dependent on recA function.

A gene required for growth and viability in recA mutants of Escherichia coli K-12 was identified. This gene, rdgB (for Rec-dependent growth), mapped near 64 min on the E. coli genetic map. In a strain carrying a temperature-sensitive recA allele, recA200, and an rdgB mutation, DNA synthesis but not protein synthesis ceased after 80 min of incubation at 42 degrees C, and there was extensive DNA degradation. The rdgB mutation alone had no apparent effect on DNA synthesis or growth; however, mutant strains did show enhanced intrachromosomal recombination and induction of the SOS regulon. The rdgB gene was cloned and its-gene product identified through the construction and analysis of deletion and insertion mutations of rdgB-containing plasmids. The ability of a plasmid to complement an rdgB recA mutant was correlated with its ability to produce a 25-kilodalton polypeptide as detected by the maxicell technique.     

Role of the recF gene of Escherichia coli K-12 in lambda recombination

Summary When Escherichia coli K12() lysogens are infected with heteroimmune phage, which are unable to replicate, general recombination between phage and prophage depends on the bacterial recF gene. It has been shown that in E. coli K12 postconjugational recombination, the RecF pathway only works with full efficiency if exonuclease I is absent (Clark 1973). However, results presented in this paper indicate that under conditions in which replication is blocked, the recombination pathway dependant on the recF gene is fully active in producing viral recombinants even, if the phage is Red⁺, in the presence of exonuclease I. In contrast, removal of exonuclease and protein requires elimination of exonuclease I for an efficient RecF pathway. It is concluded that the Red system cooperates with the RecF pathway and that this cooperation involves overcoming the inhibitory effects of exonuclease I. In the absence of exonuclease, protein stimulates recF-dependent recombination but does not suffice to prevent the negative effect of exonuclease I. In the presence of protein, full efficiency of the RecF pathway can be obtained either via cooperation with exonuclease I or, if the viral exonuclease is defective, via inactivation of exonuclease I. Since activity of exonuclease appears necessary to overcome the inhibitory effects of exonuclease I, it is proposed here that exonuclease diverts material from the RecF pathway in a shunt reaction which allows completion of recF-initiated recombinational intermediates via a mechanism insensitive to exonuclease I.When replication is allowed, the Rec system produces viral recombinants mainly via a recF-independent mechanism. However, a major contribution of the RecF pathway to recombination is observed after removal of the Red system and exonuclease I.Obra social de la Caja de Ahorros de Valencia (Director: S. Grisolía)     

RecOR suppression of recF mutant phenotypes in Escherichia coli K-12.

The recF, recO, and recR genes form the recFOR epistasis group for DNA repair. recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response. Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above. We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes. Expression of the chromosomal recO+ gene is essential for the recR+ suppression. Hence we call this RecOR suppression of recF mutant phenotypes. RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome. This is not true for RecOR suppression of UV sensitivity (the two are equal). Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+. We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair.     

Cloning, expression, and characterization of the Escherichia coli K-12 rfaD gene.

The rfaD gene encodes ADP-L-glycero-D-mannoheptose-6-epimerase, an enzyme required for the biosynthesis of the lipopolysaccharide precursor ADP-L-glycerol-D-mannoheptose. The precise localization of the rfaD gene on a 1.3-kilobase SspI-HpaI fragment is reported. The rfaD gene and the flanking regions were completely sequenced. The location of the rfaD gene on the physical map of the Escherichia coli chromosome was determined. Primer extension studies were used to define the regulatory region of the rfaD gene. The cloned rfaD gene directed the synthesis of a 37,000-dalton polypeptide in several in vivo and in vitro expression systems. N-terminal analysis of purified ADP-L-glycero-D-mannoheptose-6-epimerase confirmed the first 34-amino-acid sequence deduced from the nucleotide sequence of the rfaD gene coding region. The primary structure of the rfaD protein contains the sequence fingerprint for the ADP-binding beta alpha beta fold at the N terminus.     

Isolation of protease-proficient, recombinase-deficient recA mutants of Escherichia coli K-12

We isolated recA mutants with altered protease activity and then examined recombinase activity to determine whether the protease and recombinase functions of the RecA protein of Escherichia coli are separable. We found five mutants that had moderately strong constitutive RecA protease activity but no recombinase activity above the delta recA strain background, the first clear-cut examples of mutants of this class, designated Prtc Rec-. We also isolated 65 mutants that were protease-defective toward the LexA repressor and found that all of them were also recombinase deficient. Four of these mutants retained both partial recombinase activity and partial inducible protease activity. The recombinase-defective mutants were much more sensitive than the recA+ strain to crystal violet, kanamycin, and chloramphenicol, indicating altered membrane permeability. The recA (Prtc Rec-) mutants had a subtle alteration in protease specificity, all being defective in spontaneous induction of phages lambda imm434 and 21. They differed from Prtc Rec+ mutants of comparable or even weaker constitutive protease strength, all of which showed dramatic spontaneous induction of these prophages. However, treating a Prtc Rec- mutant with mitomycin C resulted in significant prophage induction. Thus, the RecA proteins of the Prtc Rec- mutants have constitutive protease activity toward the LexA repressor, but have only DNA damage-activable protease activity toward phage repressors. UV-induced mutagenesis from his to his+ was studied for one Prtc Rec- mutant, and induced mutation frequencies as high as those for the recA+ strain were found despite the absence of recombinase activity.     

Cloning and characterization of the dcm locus of Escherichia coli K-12.

The dcm locus of Escherichia coli K-12 has been shown to code for a methylase that methylates the second cytosine within the sequence 5'-CC(A/T)GG-3'. This sequence is also recognized by the EcoRII restriction-modification system coded by the E. coli plasmid N3. The methylase within the EcoRII system methylates the same cytosine as the dcm protein. We have isolated, from a library of E. coli K-12 DNA, two overlapping clones that carry the dcm locus. We show that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain. We also show that the cloned gene codes for a methylase, that it complements mutations in the EcoRII methylase, and that it protects EcoRII recognition sites from cleavage by the EcoRII endonuclease. We found no phage restriction activity associated with the dcm clones.     

Cloning and characterization of Escherichia coli K-12 regulator gene tyrR.

The Escherichia coli K-12 regulator gene tyrR was cloned into the multicopy plasmid pBR322 from a lambda(Tn10)tyrR+ specialized transducing phage. Further subcloning localized the gene within a 2.1-kilobase region. Analysis of plasmid-coded proteins showed that the tyrR gene codes for a 63,000-dalton polypeptide.     

Plasmidic recombination in Escherichia coli K-12: the role of recF gene function

Interplasmidic and intraplasmidic recombination proficiencies were determined in E. coli bacterial strains carrying rec mutations. Our results defined the role of recF gene function, recB, recC, and sbcB gene products (exonuclease V and exonuclease I) in plasmidic recombination in wild-type E. coli cells and in cells in which the recE recombination pathway is activated. RecF gene function is required for interplasmidic recombination regardless of the recB recC genotype. Intraplasmidic recombination is recF dependent in cells having a functional exonuclease V, but not in recB recC mutants. Exonuclease V activity inhibits both interplasmidic and intraplasmidic recombination via the recE pathway.