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PubMed1993

In vivo studies on the interaction of RecBCD enzyme and lambda Gam protein.

The interaction between the RecBCD enzyme of Escherichia coli and the lambda Gam protein was investigated. Two types of experiments were done. In one type, Gam protein was produced by transient induction of the cells lysogenic for lambda cI857gam+. The presence of Gam protein, which inhibits RecBCD nuclease, enabled these cells to support the growth of a gene 2 mutant of bacteriophage T4 (T4 2). The lysogens overproducing the RecB subunit of RecBCD enzyme could titrate Gam protein and thus prevent the growth of T4 2. In contrast, the lysogens overproducing either RecC or RecD retained their capacity for growth of T4 2. It is therefore concluded that the RecB subunit is capable of binding Gam protein. In the second type of experiments, Gam protein was provided by derepressing the gamS gene on the plasmid pSF117 (S. A. Friedman and J. B. Hays, Gene 43:255-263, 1986). The presence of this protein did not interfere with the growth of wild-type cells (which were F-). Gam protein had a certain effect on recF mutants, whose doubling time became significantly longer. This suggests that the recF gene product plays an important role in maintenance of viability of the Gam-expressing cells. Gam protein exerted the most striking effect on growth of Hfr bacteria. In its presence, Hfr bacteria grew extremely slowly, but their ability to transfer DNA to recipient cells was not affected. We showed that the effect on growth of Hfr resulted from the interaction between the RecBCD-Gam complex and the integrated F plasmid.

Bacteriophage lambda↗
PubMed1993

Genetic analysis of double-strand break repair in Escherichia coli.

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.

Bacterial Proteins↗
PubMed1993

Requirement for the polymerization and 5'-->3' exonuclease activities of DNA polymerase I in initiation of DNA replication at oriK sites in the absence of RecA in Escherichia coli rnhA mutants.

In previous studies, we found that the requirement for RecA protein in constitutive stable DNA replication (cSDR) can be bypassed by derepression of the LexA regulon and that DNA polymerase I (DNA PolI) is essential for this Rip (RecA-independent process) pathway of cSDR (Y. Cao, R. R. Rowland, and T. Kogoma, J. Bacteriol. 175:7247-7253, 1993). In this study, the role of DNA PolI in the Rip pathway was further examined. By using F' plasmids carrying different parts of the polA gene, a series of complementation tests was carried out to investigate the requirement for the three enzymatic activities, polymerization, 3'-->5' exonuclease, and 5'-->3' exonuclease activities, of DNA PolI. The result indicated that both the 5'-->3' exonuclease and polymerization activities of DNA PolI are essential for bypassing the requirement for RecA in cSDR but that the 3'-->5' exonuclease activity can be dispensed with. Complementation experiments with rat DNA Pol beta also supported the hypothesis that a nick translation activity is probably involved in cSDR in the absence of RecA. An analysis of DNA synthesis suggested that DNA PolI is involved in the initiation but not the elongation stage of cSDR. Moreover, the dnaE293(Ts) mutation was shown to render the bypass replication temperature sensitive despite the presence of active DNA PolI, suggesting that DNA PolIII is responsible for the elongation stage of the Rip pathway. A model which describes the possible roles of RecA in cSDR and the possible function of DNA PolI in the Rip pathway is proposed.

DNA Polymerase I↗
PubMed1993

Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity.

Escherichia coli strains bearing plasmids expressing phage P22 anti-RecBCD functions abc1 and abc2 were tested for the presence of recBC-like phenotypes. Abc2 induces moderate sensitivity to UV light in wild-type and recD mutant strains but severely sensitizes both recF and recJ mutants. Abc1 has little effect on UV sensitivity in wild-type or recF or recJ mutant hosts but increases the sensitivity of recD mutants to a UV dose of 20 J/m2 about 10-fold. Abc2 induces E. coli to segregate inviable cells during growth, interferes with the growth of lambda red gam chi+ and chi 0 phage (the effect is greater with chi+ phage), inhibits Chi and Chi-like activity as measured by lambda red gam crosses, and prevents SOS induction in response to nalidixic acid; Abc1 has no effect in these tests. Abc2, alone or with Abc1, does not allow the growth of lambda red gam in the presence of a P2 prophage but does not kill the P2 lysogenic host (as lambda Gam does). Finally, Abc2 inhibits conjugational recombination in wild-type cells to the level seen in recBC mutants. These data suggest that Abc2 inhibits the recombination-promoting ability of RecBCD but leaves the exonuclease functions intact.

Bacteriophage P22↗
PubMed1994

Salmonella recD mutations increase recombination in a short sequence transduction assay.

We have identified recD mutants of Salmonella typhimurium by their ability to support growth of phage P22 abc (anti-RecBCD) mutants, whose growth is prevented by normal host RecBCD function. As in Escherichia coli, the recD gene of S. typhimurium lies between the recB and argA genes at min 61 of the genetic map. Plasmids carrying the Salmonella recBCD+ genes restore ATP-dependent exonuclease V activity to an E. coli recBCD deletion mutant. The new Salmonella recD mutations (placed on this plasmid) eliminate the exonuclease activity and enable the plasmid-bearing E. coli deletion mutant to support growth of phage T4 gene 2 mutants. The Salmonella recD mutations caused a 3- to 61-fold increase in the ability of a recipient strain to inherit (by transduction) a large inserted element (MudA prophage; 38 kb). In this cross, recombination events must occur in the short (3-kb) sequences that flank the element in the 44-kb transduced fragment. The effect of the recD mutation depends on the nature of the flanking sequences and is likely to be greatest when those sequences lack a Chi site. The recD mutation appears to minimize fragment degradation and/or cause RecBC-dependent recombination events to occur closer to the ends of the transduced fragment. The effect of a recipient recD mutation was eliminated if the donor P22 phage expressed its Abc (anti-RecBC) function. We hypothesize that in standard (high multiplicity of infection) P22-mediated transduction crosses, recombination is stimulated both by Chi sequences (when present in the transduced fragment) and by the phage-encoded Abc protein which inhibits the host RecBCD exonuclease.

Bacteriophage P22↗
PubMed1994

The DNA replication fork blocked at the Ter site may be an entrance for the RecBCD enzyme into duplex DNA.

In Escherichia coli, eight kinds of chromosome-derived DNA fragments (named Hot DNA) were found to exhibit homologous recombinational hotspot activity, with the following properties. (i) The Hot activities of all Hot DNAs were enhanced extensively under RNase H-defective (rnh) conditions. (ii) Seven Hot DNAs were clustered at the DNA replication terminus region on the E. coli chromosome and had Chi activities (H. Nishitani, M. Hidaka, and T. Horiuchi, Mol. Gen. Genet. 240:307-314, 1993). Hot activities of HotA, -B, and -C, the locations of which were close to three DNA replication terminus sites, the TerB, -A, and -C sites, respectively, disappeared when terminus-binding (Tau or Tus) protein was defective, thereby suggesting that their Hot activities are termination event dependent. Other Hot groups showed termination-independent Hot activities. In addition, at least HotA activity proved to be dependent on a Chi sequence, because mutational destruction of the Chi sequence on the HotA DNA fragment resulted in disappearance of the HotA activity. The HotA activity which had disappeared was reactivated by insertion of a new, properly oriented Chi sequence at the position between the HotA DNA and the TerB site. On the basis of these observations and positional and orientational relationships between the Chi and the Ter sequences, we propose a model in which the DNA replication fork blocked at the Ter site provides an entrance for the RecBCD enzyme into duplex DNA.

Bacterial Proteins↗
PubMed1994

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.

Bacterial Proteins↗
PubMed1995

Recombinational rescue of the stalled DNA replication fork: a model based on analysis of an Escherichia coli strain with a chromosome region difficult to replicate.

To examine the physiological effects of DNA replication arrest at the terminus (Ter), we constructed a replication-blocked Escherichia coli strain so that both bidirectional replication forks would be impeded at two flanking Ter sites, one artificial and the other natural. While the blocked strain grew slightly more slowly than a control strain, it had abnormal phenotypes similar to those of E. coli dam mutants, i.e., hyper-Rec phenotype, recA(+)- and recB+ (C+)-dependent growth, and constitutive SOS induction. The observation that these two apparently unrelated mutants cause similar phenotypes led us to design a model. We propose that the following sequential events may occur in both strains. A double-strand (ds) break occurs at the blocked replication fork in the blocked strain and at the ongoing fork in the dam mutant, through which RecBCD enzyme enters and degrades the ds DNA molecule, and the degradation product serves as the signal molecule for SOS induction. When RecBCD enzyme meets an appropriately oriented Chi sequence, its DNase activity is converted to recombinase enzyme, which is able to repair the ds end, recombinationally. this model (i) explains the puzzling phenotype of recA and recB (C) mutants and the SOS-inducing phenotype of polA, lig, and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence, and (iii) suggests a possible key role for homologous recombination with regard to cell survival following the arrest of DNA replication.

Chromosomes, Bacterial↗
PubMed1996

Redundant homosexual F transfer facilitates selection-induced reversion of plasmid mutations.

F plasmids use surface exclusion to prevent the redundant entry of additional F plasmids during active growth of the host cells. This mechanism is relaxed during stationary phase and nonlethal selections, allowing homosexual redundant plasmid transfer. Homosexual redundant transfer occurs in stationary-phase liquid cultures, within nongrowing populations on solid media, and on media lacking a carbon source. We examined the relationship between homosexual redundant transfer, which occurs between F+ hosts, and reversion of a plasmid-encoded lac mutant allele, lacI33omegalacZ. Sodium dodecyl sulfate (SDS) and mutations that prevent normal transfer to F- cells reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. A recA null mutation reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ mutation. Conversely, a recD null mutation increased redundant transfer and selection-induced reversion of the lacI33omegalacZ allele. These results suggest an explanation for why SDS and these mutations affect reversion of the plasmid lacI33omegalacZ allele. However, a direct causal relationship between transfer and reversion remains to be established. These results suggest that Rec proteins play an active role in redundant transfer and/or that redundant transfer is regulated with the activation of recombination. Redundant homosexual plasmid transfer during a period of stress may represent a genetic response that facilitates evolution of plasmid-encoded functions through mutation, recombination, reassortment, and dissemination of genetic elements present in the populations.

Alleles↗
PubMed1996

Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination.

A role for the RecF, RecJ, and SbcB proteins in the RecBCD-dependent recombination pathway is suggested on the basis of the effect of null recF, recJ, and sbcB mutations in Salmonella typhimurium on a "short-homology" P22 transduction assay. The assay requires recombination within short (approximately 3-kb) sequences that flank the selected marker and lie at the ends of the transduced fragment. Since these ends are subject to exonucleolytic degradation, the assay may demand rapid recombination by requiring that the exchange be completed before the essential recombining sequences are degraded. In this assay, recF, recJ, and sbcB null mutations, tested individually, cause a small decrease in recombinant recovery but all pairwise combinations of these mutations cause a 10- to 30-fold reduction. In a recD mutant recipient, which shows increased recombination, these pairwise mutation combinations cause a 100-fold reduction in recombinant recovery. In a standard transduction assay (about 20 kb of flanking sequence), recF, recJ, and sbcB mutations have a very small effect on recombinant frequency. We suggest that these three proteins promote a rate-limiting step in the RecBC-dependent recombination process. The above results were obtained with a lysogenic recipient strain which represses expression of superinfecting phage genomes and minimizes the contribution of phage recombination functions. When a nonlysogenic recipient strain is used, coinfecting phage genomes express functions that alter the genetic requirements for recombination in the short-homology assay.

Bacterial Proteins↗
PubMed1996

Plasmid recombination by the RecBCD pathway of Escherichia coli.

Previously, we demonstrated that exonuclease I-deficient strains of Escherichia coli accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids carrying the chi sequence (5'- GCTGGTGG-3') (M. M. Zaman and T. C. Boles, J. Bacteriol. 176:5093-5100, 1994). Since high-molecular weight linear DNA is believed to be the natural substrate for RecBCD-mediated recombination during conjugation (A. J. Clark and K. B. Low, p. 155-215, in K. B. Low, ed., The Recombination of Genetic Material, 1988), we analyzed the recombination frequencies of chi+ and chi0 plasmids in sbcB strains. Here, we report that chi sites stimulate plasmid recombination frequency by 16-fold in sbcB strains. Chi-stimulated plasmid recombination is dependent on RecBCD but is independent of RecF pathway genes. The distribution of recombination products suggests that high-molecular-weight linear plasmid DNA is a substrate for RecBCD-mediated recombination. Surprisingly, our data also suggest that chi+ plasmids also recombine by the RecBCD pathway in rec+ sbcB+ cells.

Base Sequence↗
PubMed1996

The C terminus of the AddA subunit of the Bacillus subtilis ATP-dependent DNase is required for the ATP-dependent exonuclease activity but not for the helicase activity.

Comparison of subunit AddA of the Bacillus subtilis AddAB enzyme, subunit RecB of the Escherichia coli RecBCD enzyme, and subunit RecB of the Haemophilus influenzae RecBCD enzyme revealed several regions of homology. Whereas the first seven regions are common among helicases, the two C-terminally located regions are unique for RecB of E. coli and H. influenzae and AddA. Deletion of the C-terminal region resulted in the production of an enzyme which showed moderately impaired levels of ATP-dependent helicase activity, whereas the ATP-dependent exonuclease activity was completely destroyed. The mutant enzyme was almost completely capable of complementing E. coli recBCD and B. subtilis addAB strains with respect to DNA repair and homologous recombination. These results strongly suggest that at least part of the C-terminal region of the AddA protein is indispensable for exonuclease activity and that, in contrast to the exonuclease activity, the helicase activity of the addAB gene product is important for DNA repair and homologous recombination.

Amino Acid Sequence↗
PubMed1996

Role of the recJ gene product in UV-induced illegitimate recombination at the hotspot.

Illegitimate recombination between a prophage and adjacent bacterial DNA is the first step in the formation of specialized transducing phage. Such recombination is rare, but it is greatly enhanced by UV irradiation. We studied the mechanism of UV-induced illegitimate recombination by examining the effect of rec mutations on the frequency of lambda bio transducing phage and found that an Escherichia coli recJ mutation reduces it by 3- to 10-fold. In addition, the recombination hotspot, which accounts for approximately 60% of lambda bio transducing phages in wild-type bacteria, was not detected in the recJ mutant. Introduction of a RecJ overexpression plasmid into the recJ mutant recovered the recombination at the hotspot. These results indicate that the RecJ protein preferentially stimulates illegitimate recombination at the hotspot. Both the hotspot and the non- hotspot sites have short regions of homology, but only the hotspot sites contain common direct-repeat sequences. We propose a model based on the 5'-3' exonuclease activity of RecJ to explain the involvement of this protein in illegitimate recombination at the hotspot.

Bacterial Proteins↗
PubMed1997

Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum.

Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments. Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution. Here we report on the first protein to be characterized from one of these organisms. The DNA polymerase gene of C. symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig. Its deduced amino acid sequence is highly similar to those of the archaeal family B (alpha-type) DNA polymerases. It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively). The conserved motifs of B (alpha-)-type DNA polymerases and 3'-5' exonuclease were identified in the 845-amino-acid sequence. The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags. It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate. Single-strand- and double-strand-dependent 3'-5' exonuclease activity was detected, as was a marginal 5'-3' exonuclease activity. The enzyme was rapidly inactivated at temperatures higher than 40 degrees C, with a half-life of 10 min at 46 degrees C. It was found to be less thermostable than polymerase I of E. coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes. Although phylogenetic studies suggest a thermophilic ancestry for C. symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution.

Amino Acid Sequence↗
PubMed1997

Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation.

To study the fate of linear DNA in Escherichia coli cells, we linearized plasmid DNA at a specific site in vivo and monitored its behavior in recA mutant cells deficient in recombinational repair. Earlier, we had found that in wild-type (WT) cells linearized DNA is degraded to completion by RecBCD nuclease. We had also found that in WT cells chi sites on linear DNA inhibit RecBCD degradation by turning off its nucleolytic activities. Now we report that chi sites do not work in the absence of the RecA protein, suggesting that RecA is required in vivo to turn off the degradative activities of the RecBCD enzyme. We also report that the degradation of linearized plasmid DNA, even devoid of chi sites, is never complete in recA cells. Investigation of this linear DNA stability indicates that a fraction of recA cells are recBC phenocopies due to ongoing chromosomal DNA degradation, which titrates RecBCD nuclease. A possible role for RecBCD-promoted DNA degradation in controlling chromosomal DNA replication in E. coli is discussed.

Chromosomes, Bacterial↗
PubMed1997

Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis.

The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the major recombination enzyme, RecA. Strains carrying a mutation in the ATP-binding site of the AddB subunit exhibited high levels of plasmid instability, whereas a comparable mutation in the A subunit did not affect plasmid stability. Using an alternative plasmid system, pGP100, we were able to demonstrate that the differences in stability reflected differences in initial recombination frequencies. Based on a comparison of endpoint sequences observed in the various hosts, we speculate that at least two different mechanisms underlie the deletion events involved, the first (type I) occurring between nonrepeated sequences, and the second (type II) occurring between short direct repeats (DRs). The latter event was independent of single-strand replication intermediates and the mode of replication and possibly requires the introduction of double-strand breaks (DSBs) between the repeats. In the absence of functional AddAB complex, or the AddB subunit, DSBs are likely to be processed via a recA-independent mechanism, resulting in intramolecular recombination between the DRs. In wild-type cells, such DSBs are supposed to be either repaired by a mechanism involving AddAB-dependent recombination or degraded by the AddAB-associated exonuclease activity. Plasmid stability assays in a recA mutant showed that (i) the level of deletion formation was considerably higher in this host and (ii) that deletions between short DRs occurred at higher frequencies than those described previously for the parental strain. We propose that in wild-type cells, the recA gene product is involved in recombinational repair of DSBs.

Adenosine Triphosphate↗
PubMed1997

Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism.

Two novel types of alleviation of DNA restriction by the EcoKI restriction endonuclease are described. The first type depends on the presence of the gam gene product (Gam protein) of bacteriophage lambda. The efficiency of plating of unmodified phage lambda is greatly increased when the restricting Escherichia coli K-12 host carries a gam+ plasmid. The effect is particularly striking in wild-type strains and, to a lesser extent, in the presence of sbcC and recA mutations. In all cases, Gam-dependent alleviation of restriction requires active recBCD genes of the host and recombination (red) genes of the infecting phage. The enhanced capacity of Gam-expressing cells to repair DNA strand breaks might account for this phenomenon. The second type is caused by the presence of a plasmid in a restricting host lacking RecBCD enzyme. Commonly used plasmids such as the cloning vector pACYC184 can produce such an effect in strains carrying recB single mutations or in recBC sbcBC strains. Plasmid-mediated restriction alleviation in recBC sbcBC strains is independent of the host RecF, RecJ, and RecA proteins and phage recombination functions. The presence of plasmids can also relieve restriction in recD strains. This effect depends, however, on the RecA function in the host. The molecular mechanism of the plasmid-mediated restriction alleviation remains unclear.

Bacterial Proteins↗