RecF Function Research Articles

Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function

RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA-like recombinases and displacing single-stranded DNA-binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled-coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so-called 'Lobe II' subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double-stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP-dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.

ATP Hydrolysis and DNA Binding by the Escherichia coli RecF Protein

The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed.

Mechanism and control of interspecies recombination in Escherichia coli. I. Mismatch repair, methylation, recombination and replication functions.

A genetic analysis of interspecies recombination in Escherichia coli between the linear Hfr DNA from Salmonella typhimurium and the circular recipient chromosome reveals some fundamental aspects of recombination between related DNA sequences. The MutS and MutL mismatch binding proteins edit (prevent) homeologous recombination between these 16% diverged genomes by at least two distinct mechanisms. One is MutH independent and presumably acts by aborting the initiated recombination through the UvrD helicase activity. The RecBCD nuclease might contribute to this editing step, presumably by preventing reiterated initiations of recombination at a given locus. The other editing mechanism is MutH dependent, requires unmethylated GATC sequences, and probably corresponds to an incomplete long-patch mismatch repair process that does not depend on UvrD helicase activity. Insignificant effects of the Dam methylation of parental DNAs suggest that unmethylated GATC sequences involved in the MutH-dependent editing are newly synthesized in the course of recombination. This hypothetical, recombination-associated DNA synthesis involves PriA and RecF functions, which, therefore, determine the extent of MutH effect on interspecies recombination. Sequence divergence of recombining DNAs appears to limit the frequency, length, and stability of early heteroduplex intermediates, which can be stabilized, and the recombinants mature via the initiation of DNA replication.

Pathways for homologous recombination between chromosomal direct repeats in Salmonella typhimurium.

Homologous recombination pathways probably evolved primarily to accomplish chromosomal repair and the formation of and resolution of duplications by sister-chromosome exchanges. Various DNA lesions initiate these events. Classical recombination assays, involving bacterial sex, focus attention on double-strand ends of DNA. Sexual exchanges, initiated at these ends, depend on the RecBCD pathway. In the absence of RecBCD function, mutation of the sbcB and sbcC genes activates the apparently cryptic RecF pathway. To provide a more general view of recombination, we describe an assay in which endogenous DNA damage initiates recombination between chromosomal direct repeats. The repeats flank markers conferring lactose utilization (Lac+) and ampicillin resistance (ApR); recombination generates Lac-ApS segregants. In this assay, the RecF pathway is not cryptic; it plays a major role without sbcBC mutations. Others have proposed that single-strand gaps are the natural substrate for RecF-dependent recombination. Supporting this view, recombination stimulated by a double-strand break (DSB) in a chromosomal repeat depended on RecB function, not RecF function. Without RecBCD function, sbcBC mutations modified the RecF pathway and allowed it to catalyze DSB-stimulated recombination. Sexual recombination assays overestimate the importance of RecBCD and DSBs, and underestimate the importance of the RecF pathway.

Molecular characterization of the Azotobacter vinelandii recF gene

The recF gene from Azotobacter vinelandii (Av) has been cloned by complementation in an Escherichia coli (Ec) recF mutant. The sequence of 1568 bp has been determined and analyzed. It showed an open reading frame of 1092 nt coding for a 364-amino-acid (aa) polypeptide. The comparison of the deduced aa sequence of the recF of Av with those of other bacteria has elicited the presence of the four conserved domains thought to be essential for RecF function. A transcriptional fusion of a DNA fragment containing the promoter sequence of recF with the lacZ gene of Ec was constructed and 3-4-fold enhancement of promoter activity was observed upon UV induction.

Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli

Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3'. Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions. In addition, chi stimulation occurs only in cis. Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli II. Stimulation of RecF-dependent recombination by excision repair of cyclobutane pyrimidine dimers and of other photoproducts.

Three aspects of recombination of UV-irradiated nonreplicating lambda phage DNA were addressed: the photoproduct(s) responsible, the role of UvrABC-mediated excision repair, and the dependence on RecF function. Cyclobutane pyrimidine dimers appeared responsible for some recombination because photoreactivation reduced the frequency of 254-nm-stimulated recombination and because photosensitized 313-nm irradiation stimulated recombination. Other photoproducts seemed recombinogenic as well, because high fluences of 254-nm irradiation stimulated recombination considerably more, per cyclobutane dimer induced, than photosensitized 313-nm irradiation, and because photoreactivation did not eliminate 254-nm stimulated recombination. For both treatments, much, but not all, of the recombination was UvrABC-dependent. Recombination was mostly RecF-dependent, but was not affected by recB recC or recE mutations

Genetic control of damage-inducible restriction alleviation in Escherichia coli K12: an SOS function not repressed by lexA.

The alleviation of K-specific DNA restriction after treatment of cells by UV or nalidixic acid has been studied in mutants with various alleles of recA and lexA and combinations of these alleles and with recB and recF mutations. The studies show that induction of restriction alleviation by UV or nalidixic acid is abolished in mutants in which the recA protein is defective (recA13, recA56), its protease activity is altered (recA430) or in which it cannot be efficiently activated (recA142). Thermoinduction of restriction alleviation was observed in tif mutant (recA441). In lexA amber mutants restriction alleviation is not constitutive but is still inducible. In a lexA3 mutant restriction alleviation is inducible by nalidixic acid provided that recA protein is overproduced as a result of a recA operator mutation. Induction by UV depends on the recF function and an unidentified function (Y) which is controlled by the lexA protein. The recBC enzyme is necessary for induction by UV or by nalidixic acid. Temperature shift experiments with a thermosensitive recB mutant indicate that the recBC enzyme functions in an early step during UV-induction. It is concluded that the damage-inducible function which alleviates restriction is similar to other damage inducible repair (SOS) functions in the dependence on activated recA protease for induction, but that it differs from these functions by the absence of a direct control through the lexA repressor.

Suppression of Escherichia coli recF mutations by recA-linked srfA mutations

Suppressors of recF (srfA) were found by selection for resistance to mitomycin C and UV irradiation in a recB21 recC22 sbcB15 recF143 strain. srfA mutations map in recA and are dominant to srfA+. They suppress both the DNA repair and the recombination deficiencies due to recF mutations. Therefore, RecA protein which is altered by the srfA mutation can allow genetic recombination to proceed in the absence of recB, recC, and recF functions. recF is also required for induction of the SOS response after UV damage. We propose that recF+ normally functions to allow the expression of two recA activities, one that is required for the RecF pathway of recombination and another that is required for SOS induction. The two RecA activities are different and are separable by mutation since srfA mutations permit recombination to proceed but have not caused a dramatic increase in SOS induction in recF mutants. According to this hypothesis, one role for recF in DNA repair and recombination is to modulate RecA activities to allow RecA to participate in these recF-dependent processes.

RecA-independent general genetic recombination of plasmids.

The small circular genomes of several bacteriophages and a number of plasmids have been used extensively to study various aspects of the mechanism of genetic recombination. Several studies have demonstrated that the interconversion between the circular monomers and larger circular oligomers of many plasmid DNAs is mediated, at least in part, by the pathway which carries out genetic recombination in wild-type Escherichia coli1–4. The recA, recB and recC mutations block conjugation-mediated recombination in E. coli5,6. However, Clark and his co-workers7,8 isolated two mutations, sbcA and sbcB, which indirectly suppress the recombination deficiency of recB and recC mutations. These indirect suppressor mutations seem to cause recombination by other pathways which require the recA gene product as well as products of other genes such as recF, recJ and recM, which are not normally required for recombination in E. coli9,10. Here we report an investigation of the effect of recombination-deficient mutations on plasmid recombination in E. coli. We show that sbcA mutations induce a new plasmid recombination pathway that is 10–15 times more efficient than the normal wild-type recombination pathway and independent of the recA and recF functions.

Control of recA gene RNA in E. coli: Regulatory and signal genes

We have studied transcription of the recA gene of E. coli as a probe for regulatory events involved in the coupled control of DNA repair, mutagenesis, cell division, and prophage induction (“SOS funtions”). RecA mRNA production is greatly stimulated by two agents that block normal chromosomal DNA replication, ultraviolet light and nalidixic acid. This specific RNA induction is blocked by recA and dominant lexA mutations that confer ultraviolet sensitivity on the cell. A constitutive high level of RecA mRNA production results from mutations believed to inactive lexA or from the unique recA mutation tif1. These results suggest that the LexA protein is a negative regulator of the recA gene at the level of RNA synthesis, whereas RecA is a positive (“auto-regulatory”) protein. The earliest (“signal”) events leading to recA gene induction appear to involve different pathways for the two types of replication blocks studied. Mutations affecting the RecBC protein block induction of RecA mRNA by nalidixic acid, but not ultraviolet light. In contrast, a loss of RecF function markedly decreases induction by ultraviolet light, but not nalidixic acid. Thus the signal system for SOS repair seems to have alternative pathways to provide a general response to a variety of DNA-damaging agents.