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Effect of DNA sequence and structure on nuclease activity of the DexA protein of bacteriophage T4.

The bacteriophage T4 dexA gene product is required during infection of Escherichia coli strains carrying a mutation in the optA gene. We purified the DexA protein from cells which overproduced the protein. The protein was assayed for nuclease activity on synthetic di- and oligonucleotide substrates of known sequence and secondary structure. Sequence and structure significantly affected nuclease activity. The properties of the enzyme may explain the requirement for the DexA protein during infection of optA mutant hosts.

Base Sequence↗

Inhibition of the SOS response of Escherichia coli by the Ada protein.

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.

Alkylation↗

Cloning of the recB, recC, and recD genes from Proteus mirabilis in Escherichia coli: in vivo formation of active hybrid enzymes.

We cloned chromosomal DNA fragments from Proteus mirabilis which complement recBCD deletion mutants of Escherichia coli by restoring (i) recombination proficiency in conjugation, (ii) normal resistance to UV irradiation, and (iii) ATP-dependent exonuclease activity for duplex DNA. The data indicate that the order of the genes thyA, recC, recB, recD, and argA is similar in both P. mirabilis and E. coli. Hybrid enzymes formed in vivo were active in repair and recombination.

Bacterial Proteins↗

Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions.

Plasmids that express the bacteriophage lambda gam gene or the P22 abc2 gene (with and without abc1) at controllable levels were placed in Escherichia coli and tested for effects on the activity of RecBCD. Like Gam, Abc2 inhibited the ATP-dependent exonuclease activity of RecBCD, apparently not by binding to DNA. However, Abc2-mediated inhibition was partial, while Gam-mediated inhibition was complete. Both Abc2 and Gam inhibited host system-mediated homologous recombination in a Chi-containing interval in the chromosome of a hybrid lambda phage; Abc2 inhibited it more strongly than Gam. Gam but not Abc2 spared a phage T4 gene 2 mutant from restriction by RecBCD; Abc2 exhibited weak sparing activity in combination with Abc1 and substantial activity in combination with both Abc1 and P22 homologous recombination function Erf. Either Gam or the combination of the lambda recombination functions Exo and Bet was sufficient to induce a mode of plasmid replication that produced linear multimers. The combination of Abc2, Abc1, and Erf also exhibited this activity. However, Erf was inactive, both by itself and in combination with Abc1; Abc2 had weak activity. These results indicate that Gam and Abc2 modulate the activity of RecBCD in significantly different ways. In comparison with lambda Gam, P22 Abc2 has a weak effect on RecBCD nuclease activity but a strong effect on its recombination-promoting activity.

Bacteriophage lambda↗

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.

Escherichia coli↗

recB and recC genes of Salmonella typhimurium.

We have investigated the genetic organization of the recB (exonuclease V) and recC (exonuclease V) genes of Salmonella typhimurium. A detailed genetic map is constructed that includes the relative order in the chromosome, P22 cotransduction frequencies, and the orientation of transcription of the recB and recC genes. In addition, the isolation and characterization of Mu dJ insertion mutations in recB and recC are discussed.

Chromosome Mapping↗

Chromosomal transformation of Escherichia coli recD strains with linearized plasmids.

Wild-type Escherichia coli are resistant to genetic transformation by purified linear DNA, probably in part because of exonuclease activity. We demonstrate that E. coli containing a recD mutation could be easily transformed by linearized plasmids containing a selectable marker. The marker was transferred to the chromosome by homologous recombination, whereas plasmid markers not in the region of homology were lost.

Bacterial Proteins↗

recB recC-dependent processing of heteroduplex DNA stimulates recombination of an adjacent gene in Escherichia coli.

The effect of DNA mismatched repair on the genetic recombination of a gene adjacent to the mismatch site (MS) was tested by using four mismatch configurations. An MS was constructed in a well-characterized plasmid recombination substrate, and recombination with a resident compatible plasmid was measured after transformation of the mismatched plasmid into Escherichia coli. The mismatched plasmids were constructed such that one of the DNA strands was methylated by the DNA adenine methylase (Dam), while the other strand was unmethylated. The processing of a hemimethylated single-base-pair mismatch had no effect on the recombination of the adjacent gene, suggesting that the most efficient (Dam-instructed) mismatch repair process does not secondarily promote genetic recombination. However, mismatches that could form an ordered secondary structure resembling a cruciform increased the recombination of this adjacent gene at least 20-fold. An identical mismatch that could not form an ordered secondary structure had no effect in this system. The increased frequency of recombination observed was found to require the recB or recC gene product or both. Furthermore, the recombination appeared unidirectional, in that the cruciform-containing plasmid did not produce stable transformants. Our results support a model in which the cruciform-containing plasmid can participate in recombination with the resident plasmid but is unable to produce stable transformant progeny. A proposed role for the RecBCD enzyme (ExoV) in this process is discussed.

DNA Repair↗

Neurospora endo-exonuclease is immunochemically related to the recC gene product of Escherichia coli.

Immunochemical cross-reaction between the endo-exonuclease of Neurospora crassa, an enzyme previously implicated in recombination and recombinational DNA repair, and the recC-encoded polypeptide of Escherichia coli has been detected by immunoblotting extracts of strains of E. coli having a deletion that includes the recBCD genes but carrying multicopy plasmids bearing all three of the recBCD genes or only one or two of these genes. It was predicted that homology would also be found at the amino acid sequence level between the recC polypeptide and both nuclear and mitochondrial endo-exonucleases of Saccharomyces cerevisiae, which cross-react with antibodies raised to the N. crassa endo-exonuclease. Since the gene for the S. cerevisiae mitochondrial enzyme, NUC1, has been cloned and sequenced and the predicted amino acid sequence is known, this sequence was aligned with the predicted amino acid sequence of the recC polypeptide. Extensive homology was found by aligning 306 of the 329 amino acids of the yeast mitochondrial nuclease sequence with the carboxy-terminal one-quarter of the amino acid sequence of the recC polypeptide.

Amino Acid Sequence↗

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 Replication↗

Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis.

The genes encoding the subunits of the Bacillus subtilis ATP-dependent nuclease (add genes) have been cloned. The genes were located on an 8.8-kb SalI-SmaI chromosomal DNA fragment. Transformants of a recBCD deletion mutant of Escherichia coli with plasmid pGV1 carrying this DNA fragment showed ATP-dependent nuclease activity. Three open reading frames were identified on the 8.8-kb SalI-SmaI fragment, which could encode three proteins with molecular masses of 135 (AddB protein), 141 (AddA protein), and 28 kDa. Only the AddB and AddA proteins are required for ATP-dependent exonuclease activity. Both the AddB and AddA proteins contained a conserved amino acid sequence for ATP binding. In the AddA protein, a number of small regions were present showing a high degree of sequence similarity with regions in the E. coli RecB protein. The AddA protein contained six conserved motifs which were also present in the E. coli helicase II (UvrD protein) and the Rep helicase, suggesting that these motifs are involved in the DNA unwinding activity of the enzyme. When linked to the T7 promoter, a high level of expression was obtained in E. coli.

Amino Acid Sequence↗

Linkage distortion following conjugational transfer of sbcC+ to recBC sbcBC strains of Escherichia coli.

Conjugational recombination in Escherichia coli depends normally on RecBCD enzyme, a multifunctional nuclease and DNA helicase produced by the recB, recC, and recD genes. However, recombination can proceed efficiently without RecBCD in recB or recC strains carrying additional mutations in both the sbcB and sbcC genes. Recombination in these strains, sometimes referred to as the RecF pathway, requires gene products that are not essential in the RecBCD-dependent process predominating in the wild type. It has also been reported to produce a different spectrum of recombinant genotypes in crosses with Hfr donors. However, the sbcC+ gene was unknowingly transferred to the recipient strain in some of these crosses, and this may have affected the outcome. This possibility was examined by conducting parallel crosses with Hfr donors that were either wild type or mutant for sbcC. Transfer of sbcC+ from an Hfr donor is shown to alter the frequency of recombinant genotypes recovered. There is a severe reduction in progeny that inherit donor markers linked to the sbcC+ allele and an increase in the incidence of multiple exchanges. Colonies of mixed genotype for one or more of the unselected proximal markers are also much more prevalent. Since the yield of recombinants is lower than normal, these changes are attributed to the reduced viability of recombinants that inherit sbcC+ from the Hfr donor. When the Hfr donor used is also mutant for sbcC, the yield of recombinants is greater and the frequencies of the different genotypes recovered are similar to those obtained in crosses with a rec+ sbc+ recipient, in which transfer of sbcC+ has no apparent effect. Earlier studies are re-examined in light of these findings. It is concluded that, while recombination in recBC sbcBC strains involves different enzymes, the underlying molecular mechanism is essentially the same as that in the wild type.

Chromosomes, Bacterial↗

Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme.

The lambda Gam protein was isolated from cells containing a Gam-producing plasmid. The purified Gam protein was found to bind to RecBCD without displacing any of its subunits. Gam was shown to inhibit all known enzymatic activities of RecBCD: ATP-dependent single- and double-stranded DNA exonucleases, ATP-independent single-stranded endonuclease, and the ATP-dependent helicase. When produced in vivo, Gam inhibited chi-activated recombination in lambda red gam crosses but had little effect on the host's ability to act as a recipient in conjugational recombination. These experiments suggest that RecBCD possesses an additional "unknown" activity that is resistant to or induced by Gam. Additionally, the expression of Gam in recD mutants sensitizes the host to UV irradiation, indicating that Gam alters one or more of the in vivo activities of RecBC(D-).

Amino Acid Sequence↗

Purification and characterization of DNA polymerases from Bacillus species.

DNA polymerases from Bacillus stearothermophilus, Bacillus caldotenax, and Bacillus caldovelox were purified by chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose and obtained in high yield. The enzyme preparations are free of exo- and endonuclease activities. Additional purification steps, e.g., hydrophobic interaction chromatography and chromatography on a Mono Q column or sucrose density gradient centrifugation, are needed to obtain the enzymes in the form of homogeneous 95-kDa proteins. Each of the three organisms possesses a major DNA polymerase activity comparable to DNA polymerase I. The enzymes require Mg2+ (10 to 30 mM) for optimal activity, although 0.4 mM Mn2+ could substitute for magnesium. The optimal reaction temperatures were lowest in B. stearothermophilus (60 to 65 degrees C) and about equal in B. caldovelox and B. caldotenax (65 to 70 degrees C). The thermal stabilities of the enzymes increased in the same order. The DNA polymerase from Thermus thermophilus was isolated for comparison by using a similar procedure. The enzyme was obtained as a homogeneous 85-kDa protein that was also free of exo- and endonucleolytic activities.

Bacillus↗

Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding.

Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.

DNA Helicases↗

Efficiency of homologous intermolecular recombination at different locations on the Bacillus subtilis chromosome.

The efficiencies of intermolecular recombination at 12 different locations on the Bacillus subtilis chromosome were determined by transforming competent cells with a nonreplicative plasmid. The efficiencies varied by only about threefold but were significantly different (P less than 0.05 by a chi-square test) for approximately 20% of the locations. The recA gene product is required for recombination, and the addA gene product appears to affect the variation in a site-specific way.

Bacillus subtilis↗

Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli.

Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.

DNA Helicases↗

Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12.

The function of an open reading frame (orf-45) located upstream of the sbcC gene of Escherichia coli was investigated. Mutations that inactivate sbcC improve the ability to propagate lambda red gam phage that carry a palindromic sequence in their DNA. They also act with sbcB mutations as cosuppressors of the defects in recombination, DNA repair, and cell viability associated with recBC mutations. A 1,282-bp cassette encoding resistance to kanamycin was used to disrupt orf-45. The mutation, which has a polar effect on the expression of sbcC, allowed stable propagation of palindromic lambda phage even when the sbcC gene product was provided in trans. Additional nonpolar mutations in orf-45 were isolated on the basis of their ability to improve the growth of recBC sbcB strains. These mutations also confer resistance to mitomycin C, allow efficient recombination in Hfr crosses, and facilitate stable propagation of palindromic phage. It is concluded that the products of orf-45 and sbcC are functionally related. The orf-45 gene is therefore renamed sbcD.

Bacteriophage lambda↗