recC Mutants Research Articles

RecBCD Enzyme "Chi Recognition" Mutants Recognize Chi Recombination Hotspots in the Right DNA Context.

RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 5' GCTGGTGG 3'. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage λ. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 5' GCTGGTGG 3'. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain.

The hybrid recombinational repair pathway operates in a χ activity deficient recC1004 mutant of Escherichia coli

Homologous recombination is a crucial process for the maintenance of genome integrity. The two main recombination pathways in Escherichia coli (RecBCD and RecF) differ in the initiation of recombination. The RecBCD enzyme is the only component of the RecBCD pathway which acts in the initiation of recombination, and possesses all biochemical activities (helicase, 5'-3' exonuclease, χ cutting and loading of the RecA protein onto single-stranded (ss) DNA) needed for the processing of double stranded (ds) DNA breaks (DSB). When the nuclease and RecA loading activities of the RecBCD enzyme are inactivated, the proteins of the RecF recombination machinery, i.e., RecJ and RecFOR substitute for the missing 5'-3' exonuclease and RecA loading activity respectively. The above mentioned activities of the RecBCD enzyme are regulated by an octameric sequence known as the χ site (5'-GCTGGTGG-3'). One class of recC mutations, designated recC*, leads to reduced χ cutting invitro. The recC1004 strain (a member of the recC* mutant class) is recombination proficient and resistant to UV radiation. In this paper, we studied the effects of mutations in RecF pathway genes on DNA repair (after UV and γ radiation) and on conjugational recombination in recC1004 and recC1004 recD backgrounds. We found that DNA repair after UV and γ radiation in the recC1004 and recC1004 recD backgrounds depends on recFOR and recJ gene products. We also showed that the recC1004 mutant has reduced survival after γ radiation. This phenotype is suppressed by the recD mutation which abolishes the RecBCD dependent nuclease activity. Finally, the genetic requirements for conjugational recombination differ from those for DNA repair. Conjugational recombination in recC1004 recD mutants is dependent on the recJ gene product. Our results emphasize the importance of the canonical χ recognition activity in DSB repair and the significance of interchange between the components of two recombination machineries in achieving efficient DNA repair.

Chi hotspot activity in Escherichia coli without RecBCD exonuclease activity: implications for the mechanism of recombination.

The major pathway of genetic recombination and DNA break repair in Escherichia coli requires RecBCD enzyme, a complex nuclease and DNA helicase regulated by Chi sites (5'-GCTGGTGG-3'). During its unwinding of DNA containing Chi, purified RecBCD enzyme has two alternative nucleolytic reactions, depending on the reaction conditions: simple nicking of the Chi-containing strand at Chi or switching of nucleolytic degradation from the Chi-containing strand to its complement at Chi. We describe a set of recC mutants with a novel intracellular phenotype: retention of Chi hotspot activity in genetic crosses but loss of detectable nucleolytic degradation as judged by the growth of mutant T4 and lambda phages and by assay of cell-free extracts. We conclude that RecBCD enzyme's nucleolytic degradation of DNA is not necessary for intracellular Chi hotspot activity and that nicking of DNA by RecBCD enzyme at Chi is sufficient. We discuss the bearing of these results on current models of RecBCD pathway recombination.

Repair by recombination of DNA containing a palindromic sequence.

We report here that homologous recombination functions are required for the viability of Escherichia coli cells maintaining a 240 bp chromosomal inverted repeat (palindromic) sequence. Wild-type cells can successfully replicate this palindrome but recA, recB or recC mutants carrying the palindrome are unviable. The dependence on homologous recombination for cell viability is overcome in sbcC mutants. Directly repeated copies of the DNA containing the palindrome are rapidly resolved to single copies in wild-type cells but not in sbcC mutants. Our results suggest that double-strand breaks introduced at the palindromic DNA sequence by the SbcCD nuclease are repaired by homologous recombination. The repair is conservative and the palindrome is retained in the repaired chromosome. We conclude that SbcCD can attack secondary structures but that repair conserves the DNA sequence with the potential to fold.

DNA structures generated during recombination initiated by mismatch repair of UV-irradiated nonreplicating phage DNA in Escherichia coli: requirements for helicase, exonucleases, and RecF and RecBCD functions.

During infection of homoimmune Escherichia coli lysogens ("repressed infections"), undamaged nonreplicating lambda phage DNA circles undergo very little recombination. Prior UV irradiation of phages dramatically elevates recombinant frequencies, even in bacteria deficient in UvrABC-mediated excision repair. We previously reported that 80-90% of this UvrABC-independent recombination required MutHLS function and unmethylated d(GATC) sites, two hallmarks of methyl-directed mismatch repair. We now find that deficiencies in other mismatch-repair activities--UvrD helicase, exonuclease I, exonuclease VII, RecJ exonuclease--drastically reduce recombination. These effects of exonuclease deficiencies on recombination are greater than previously observed effects on mispair-provoked excision in vitro. This suggests that the exonucleases also play other roles in generation and processing of recombinagenic DNA structures. Even though dsDNA breaks are thought to be highly recombinagenic, 60% of intracellular UV-irradiated phage DNA extracted from bacteria in which recombination is low--UvrD-, ExoI-, ExoVII-, or Rec(J-)--displays (near-)blunt-ended dsDNA ends (RecBCD-sensitive when deproteinized). In contrast, only bacteria showing high recombination (Mut+ UvrD+ Exo+) generate single-stranded regions in nonreplicating UV-irradiated DNA. Both recF and recB recC mutations strikingly reduce recombination (almost as much as a recF recB recC triple mutation), suggesting critical requirements for both RecF and RecBCD activity. The mismatch repair system may thus process UV-irradiated DNA so as to initiate more than one recombination pathway.

RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1 drd-19

Plasmid R1 drd-19 markedly improves the recombination deficiency of recB and recB recC mutants of Escherichia coli K12 as measured by Hfr crosses and increases their resistance to uv inactivation. The effect correlates with the production of an ATP-dependent ds DNA exonuclease in recB/R1 drd-19 cells. This paper further investigates the suppressive effect of plasmid R1 drd-19 on the recB mutation of E. coli. The gene(s) responsible for the effect was localized to the 13.1-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of R1 drd-19. The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways. It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of superoligomerization of the tester pACYC184 plasmid in recB/R1 drd-19 cells and two inter- and intramolecular plasmid recombination test systems. It is probably not inhibited by λ Gam protein and does not restrict plating of T4gp2 mutant. No significant homology between the E. coli chromosomal fragment carrying recB recC recD genes and the EcoRI-C fragment of R1 drd-19 was observed. It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid). RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway.

Mechanisms of deletion formation in Escherichin coli plasmids

A set of plasmids containing 42, 21 and 31 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 bp direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.

Interspecific recombination between Escherichia coli and Salmonella typhimurium occurs by the RecABCD pathway

Interspecific recombination in conjugation between Escherichia coli and Salmonella typhimurium is several orders of magnitude lower than intraspecies recombination and is dependent on the RecA function. This low efficiency is due to a 20% divergence in the DNA sequence. The methyl-directed ( mut H, L, S dependent) mismatch repair system appears to control the fidelity of homologous recombination; inactivating one of the Mut functions increases the interspecies recombination at least by 10 3-fold. The interspecific recombination in mutS or mutL mutant is only ≈ 10 3-fold lower than recombination in homospecific crosses as found after correction for the efficiency of mating and DNA transfer by zygotic induction experiments. The interspecific recombination is dependent on the RecABCD pathway: it was abolished in a recA mutant and decreased ≈ 10 3-fold in a recC mutant.

Requirement of RecBC enzyme and an elevated level of activated RecA for induced stable DNA replication in Escherichia coli

During SOS induction, Escherichia coli cells acquire the ability to replicate DNA in the absence of protein synthesis, i.e., induced stable DNA replication (iSDR). Initiation of iSDR can occur in the absence of transcription and DnaA protein activity, which are both required for initiation of normal DNA replication at the origin of replication, oriC. In this study we examined the requirement of recB, recC, and recA for the induction and maintenance of iSDR. We found that recB and recC mutations blocked the induction of iSDR by UV irradiation and nalidixic acid treatment. In recB(Ts) strains, iSDR activity induced at 30 degrees C was inhibited by subsequent incubation at 42 degrees C. In addition, iSDR that was induced after heat activation of the RecA441 protein was abolished by the recB21 mutation. These results indicated that the RecBC enzyme was essential not only for SOS signal generation but also for the reinitiation of DNA synthesis following DNA damage. recAo(Con) lexA3(Ind-) strains were found to be capable of iSDR after nalidixic acid treatment, indicating that the derepression of the recA gene and the activation of the elevated level of RecA protein were the necessary and sufficient conditions for the induction of iSDR.

DNA breakdown by the 4-quinolones and its significance

DNA breakdown occurred in Escherichia coli KL16 exposed to nalidixic acid, ciprofloxacin or norfloxacin. However DNA breakdown does not seem to be the cause of the lethality of the 4-quinolones because it still occurred under conditions which abolished the lethality of nalidixic acid. Furthermore, no correlation was found between the amount of DNA breakdown and the rate of death of bacteria caused by the three 4-quinolones. Similarly, DNA breakdown did not occur when recB or recC mutants were treated with nalidixic acid despite both mutants being killed by the drug, again suggesting that DNA breakdown is not the cause of bacterial death. Since recB and recC mutants lack exonuclease V, this enzyme may be responsible for the DNA breakdown observed in bacteria treated with 4-quinolones.

The P2 phage old gene: sequence, transcription and translational control

The old ( overcoming lysogenization defect) gene product of bacteriophage P2 kills Escherichia coli recB and recC mutants and interferes with phage λ growth [ Sironi et al., Virology 46 (1971) 387–396 ; Lindahl et al., Proc. Natl. Acad. Sci. USA 66 (1970) 587–594]. Specialized transducing λ phages, which lack the recombination region, can be selected by plating λ stocks on E. coli that carry the old gene on a prophage or plasmid [Finkel et al., Gene 46 (1986) 65–69]. Deletion and sequence analyses indicate that the old-encoded protein has an M r of 65 373 and that its transcription is leftward. Primer extension analyses locate the transcription start point near the right end of the virion DNA. A bacterial mutant, named pin3 and able to suppress the effects of the old gene, has been isolated [Ghisotti et al., J. Virol. 48 (1983) 616–626]. In a pin3 mutant strain, carrying the old gene on a prophage or plasmid, the amount of old transcript is greatly reduced. The effect of the pin3 mutation is abolished by the wild-type allele of argU, an arginine tRNA that reads the rare Arg codons AGA and AGG, which are used for eight of the 14 Arg codons in the old gene. Thus the pin3 allele probably stalls translation of the old mRNA, causing this mRNA to be degraded. Isoelectric focusing and electrophoretic analysis identify the old gene product as a basic protein of approx. 65 kDa.

Рестрикционный анализ и локализация фрагмента хромосомы Bacillus subtilis, частично супрессирующего мутации в генах recB recC Escherichia coli

Рестрикционный анализ и локализация фрагмента хромосомы Bacillus subtilis, частично супрессирующего мутации в генах recB recC Escherichia coli

Identification and genetic mapping of the structural gene for an essential Escherichia coli membrane protein.

Attempts to isolate conditionally lethal recB and recC mutations of Escherichia coli K-12 by P1 localized mutagenesis led to the identification of the structural gene for an essential membrane protein. Located on a 1.5-kilobase-pair DNA fragment which physically mapped immediately 5' to the thyA gene, the product of the umpA (unidentified membrane protein) gene is a 25,000 Mr membrane-associated polypeptide. These results provide an explanation for why several research groups have been unable to obtain chromosomal deletions of the entire thyA gene. A possible interaction between the umpA and thyA genes is also discussed.

Activation of recF-dependent recombination in Escherichia coli by bacteriophage lambda- and P22-encoded functions.

Escherichia coli strains bearing wild-type and mutant alleles of various recombination genes, as well as plasmids that express recombination-related genes of bacteriophages lambda and P22, were tested for their proficiency as recipients in Hfr-mediated conjugation. It was found that the homologous recombination systems of both phages could promote recombination in a recB recC mutant host. In addition, the Abc function of P22, but not the Gam function of lambda, was found to inhibit recombination in a wild-type host; however, both Abc and Gam inhibited recombination in a recF mutant host. These observations are interpreted as indicating that the recombination systems of both phages, as well as the RecBCD-modulating functions Abc and Gam, all activate the RecF recombination pathway of E. coli.

Post-replication repair and recombination in uvrA umuC strains of Escherichia coli are enhanced by vanillin, an antimutagenic compound

Effects of vanillin on UV killing of umuC mutant strains of E. coli were investigated in order to analyze the antimutagenic role of vanillin in mutagenesis. UV-irradiated uvrA umuC cells showed higher survival when plated on medium containing vanillin rather than medium without vanillin. This increased survival associated with exposure to vanillin was observed more clearly in uvrA umuC lexA(ind −) and uvrA umuC recF strains. However, the effect was inhibited by additional recB recC mutations and completely blocked by an additional recA mutation. As far as tested the increased survival of UV-treated cells by vanillin was dependent on a capacity for genetic recombination. The effect of vanillin on recombination frequency between 2 plasmid DNA, pATH4 (Cm r Tc s) and pBMX7 (Ap r Tc s), in a uvrA umuC background was investigated. A significantly higher frequency of plasmid recombination was observed when vanillin was present in the culture medium. These findings suggest that the antimutagenic effect of vanillin is the result of enhancement of a recA-dependent, error-free, pathway of post-replication repair.

Physical and biochemical characterization of cloned sbcB and xonA mutations from Escherichia coli K-12.

In Escherichia coli K-12, sbcB/xonA is the structural gene for exonuclease I, an enzyme that hydrolyzes single-stranded DNA to mononucleotides in the 3'-to-5' direction. This enzyme has been implicated in the DNA repair and recombination pathways mediated by the recB and recC gene products (exonuclease V). We have cloned several sbcB/xonA mutant alleles in bacterial plasmids and have partially characterized the cloned genes and their protein products. Two of the mutations (xonA2 and xonA6) retain no detectable exonucleolytic activity on single-stranded DNA. The xonA6 allele was shown to harbor an insertion of an IS30-related genetic element near the 3' end of the gene. Two other mutations, sbcB15 and xonA8, exhibited significantly reduced levels of exonuclease I activity as compared to the cloned wild-type gene. A correlation was observed between levels of exonuclease I activity and the ability of the sbcB/xonA mutations to suppress UV sensitivity in recB and recC strains. Also, recombinant plasmids bearing either the sbcB15 or xonA6 allele exhibited a high degree of instability during growth of their bacterial hosts. The results suggest that the sbcB/xonA gene product is a bi- or multifunctional protein that interacts with single-stranded DNA and possibly with other proteins in the suppression of genetic recombination and DNA-repair deficiencies in recB and recC mutants.

ATP-stimulated polymerase activity involving DNA polymerase I and a recB-dependent factor in extracts of Escherichia coli cells.

ATP-stimulated DNA polymerase activity involving DNA polymerase I has been found to be present in cell extracts from wild type and recC mutant strains of Escherichia coli, but not in extracts from recB strain. The activity has been separated from recBC DNase by DEAE-cellulose ion exchange. It is suggested that recB-dependent factor is involved in the ATP-stimulation of polymerase. Evidence is provided that this stimulation may be due to the interaction of recB-dependent factor with DNA polymerase I.

Synthesis of linear plasmid multimers in Escherichia coli K-12

Linear plasmid multimers were identified in extracts of recB21 recC22 strains containing derivatives of the ColE1-type plasmids pACYC184 and pBR322. A mutation in sbcB increases the proportion of plasmid DNA as linear multimers. A model to explain this is based on proposed roles of RecBC enzyme and SbcB enzyme (DNA exonuclease I) in preventing two types of rolling-circle DNA synthesis. Support for this hypothesis was obtained by derepressing synthesis of an inhibitor of RecBC enzyme and observing a difference in control of linear multimer synthesis and monomer circle replication. Reinitiation of rolling-circle DNA synthesis was proposed to occur by recA+-dependent and recA+-independent recombination events involving linear multimers. The presence of linear plasmid multimers in recB and recC mutants sheds new light on plasmid recombination frequencies in various mutant strains.

Selection of lambda Spi − transducing phages using the P2 old gene cloned onto a plasmid

The old gene product of the P2 prophage interferes with plaque formation by λ wild type phage but allows λ phages whose red and gam genes have been deleted to form small, visible plaques (the λ Spi − phenotype). The old gene product also kills Escherichia coli recB or recC mutants. We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both λSpi + and λASpi − phages. We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSClOl and found that λSpi − phages can be selected on strains that carry this plasmid. The plasmid-bome old gene kills E. coli recB mutants, providing a selection for old − mutants.

Mechanism of sbcB-suppression of the recBC-deficiency in postreplication repair in UV-irradiated Escherichia coli K-12.

The mechanism by which an sbcB mutation suppresses the deficiency in postreplication repair shown by recB recC mutants of Escherichia coli was studied. The presence of an sbcB mutation in uvrA recB recC cells increased their resistance to UV radiation. This enhanced resistance was not due to a suppression of the minor deficiency in the repair of DNA daughter-strand gaps or to an inhibition of the production of DNA double-strand breaks in UV-irradiated uvrA recB recC cells; rather, the presence of an sbcB mutation enabled uvrA recB recC cells to carry out the repair of DNA double-strand breaks. In the uvrA recB recC sbcB background, a mutation at recF produced a huge sensitization to UV radiation, and it rendered cells deficient in the repair of both DNA daughter-strand gaps and DNA double-strand breaks. Thus, an additional sbcB mutation in uvrA recB recC cells restored their ability to perform the repair of DNA double-strand breaks, but the further addition of a recF mutation blocked this repair capacity.