The RecBCD enzyme of Escherichia coli promotes recombination preferentially at X nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of X and has no nucleolytic activity. Employing phage A red gam crosses, phage T4 2-survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear X-containing DNA (produced by rolling circle replication of a plasmid with X or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for -2 h. It is concluded that RecBCD is converted into RecBC during interaction with X by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a X-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored x-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that X sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis-and trans-acting stimulators of RecBC-dependent recombination.The RecBCD enzyme is an essential component of the main homologous recombination pathway in Escherichia coli (see refs. 1 and 2). This pathway recombines linear DNA molecules during conjugation, transduction, and vegetative phage crosses and is required for repair of double-strand breaks (3, 4). RecBCD enzyme is an ATP-dependent exonuclease (exoV) that consists of three protein subunits encoded by the recB, recC, and recD genes. ExoV activity is composed of ATPdependent DNA helicase and single-stranded DNA endonuclease activities of RecBCD enzyme acting on linear doublestranded DNA to produce single-stranded DNA oligonucleotides (see ref. 5). In cells and in cell-free extracts with ATP, RecBCD provides the major linear duplex DNA-degrading activity (6-8).X hot spots of RecBCD-dependent recombination are present in the E. coli genome -1000 times and stimulate, when present in phage A, recombination in A red gam crosses with decreasing efficiency in increasing distance on the left side of their sequence 5'-GCTGGTGG-3' as written here (see ref. 9). Hot spot activity is exerted only when RecBCD enters the DNA molecule from a double-stranded end placed on the 3' side of X (see ref. 9). An important finding was that recD null mutants were recombination proficient and as UV-resistant as wild-type cells (10, 11). They had lost all nucleolytic activities of RecBCD, and recombination was x independent (10, 11).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Moreo...
We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease.
To assess the contributions of single-strand DNases (ssDNases) to recombination in a recBCD ؉ background, we studied 31 strains with all combinations of null alleles of exonuclease I (⌬xon), exonuclease VII (xseA), RecJ DNase (recJ), and SbcCD DNase (sbcCD) and exonuclease I mutant alleles xonA2 and sbcB15. The xse recJ sbcCD ⌬xon and xse recJ sbcCD sbcB15 quadruple mutants were cold sensitive, while the quadruple mutant with xonA2 was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. The absence of ssDNases hardly affected P1 transductional recombinant formation, and conjugational recombinant production was decreased (as much as 94%) in several cases. Strains with sbcB15 were generally like the wild type. We determined that the sbcB15 mutation caused an A183V exchange in exonuclease motif III and identified xonA2 as a stop codon eliminating the terminal 8 amino acids. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of wild-type Xon (Xon ؉ ), respectively, with altered activity profiles. In gel shift assays, SbcB15 associated relatively stably with 3 DNA overhangs, giving protection against Xon ؉ . In addition to their postsynaptic roles in the RecBCD pathway, exonuclease I and RecJ are proposed to have presynaptic roles of DNA end blunting. Blunting may be specifically required during conjugation to make DNAs with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein, known to activate the RecF pathway in recBC strains, contributes independently of RecF to recombination in recBCD ؉ cells. DNA end binding by SbcB15 can also explain other specific phenotypes of strains with sbcB15.The heterotrimeric RecBCD enzyme (encoded by the recB, recC, and recD genes) is a central component of the main pathway of genetic recombination and recombinational DNA repair of Escherichia coli (the RecBCD pathway) and functions in the initiation of these processes (31,34,37). The enzyme (also termed exonuclease V [ExoV]), with its DNase and helicase activities, processively degrades duplex DNA from an end until it reaches an octanucleotide termed Chi, which is present in the E. coli genome once per 5,000 nucleotides on average. Upon contact with Chi, the duplex DNA degradation activity of the enzyme is attenuated and switched to produce a 3Ј single-stranded (ss) DNA end on which it loads RecA protein, making a nucleoprotein filament ready to initiate recombination. recB or recC null mutations drastically reduce homologous recombination, increase the sensitivity of cells to DNA-damaging agents, and impair cell viability (34). In several studies extragenic suppressors of the severe effects of recBC mutations have been isolated and characterized. One group, termed sbcA mutations, was found to express recombination genes of the cryptic Rac prophage (43,68). The other group of mutations was located in the gene for ExoI and affected ExoI activity (32,33). ExoI is a 3Ј-specific processive exonuclease for ...
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.
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