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339 records · Page 19Linked to original sources
PubMed2005

NinR- and red-mediated phage-prophage marker rescue recombination in Escherichia coli: recovery of a nonhomologous immlambda DNA segment by infecting lambdaimm434 phages.

We examined the requirement of lambda recombination functions for marker rescue of cryptic prophage genes within the Escherichia coli chromosome. We infected lysogenic host cells with lambdaimm434 phages and selected for recombinant immlambda phages that had exchanged the imm434 region of the infecting phage for the heterologous 2.6-kb immlambda region from the prophage. Phage-encoded activity, provided by either Red or NinR functions, was required for the substitution. Red(-) phages with DeltaNinR, internal NinR deletions of rap-ninH, or orf-ninC were 117-, 12-, and 5-fold reduced for immlambda rescue in a Rec(+) host, suggesting the participation of several NinR activities. RecA was essential for NinR-dependent immlambda rescue, but had slight influence on Red-dependent rescue. The host recombination activities RecBCD, RecJ, and RecQ participated in NinR-dependent recombination while they served to inhibit Red-mediated immlambda rescue. The opposite effects of several host functions toward NinR- and Red-dependent immlambda rescue explains why the independent pathways were not additive in a Rec(+) host and why the NinR-dependent pathway appeared dominant. We measured the influence of the host recombination functions and DnaB on the appearance of orilambda-dependent replication initiation and whether orilambda replication initiation was required for immlambda marker rescue.

Bacteriophage lambda↗
PubMed2005

The RuvAB branch migration translocase and RecU Holliday junction resolvase are required for double-stranded DNA break repair in Bacillus subtilis.

In models of Escherichia coli recombination and DNA repair, the RuvABC complex directs the branch migration and resolution of Holliday junction DNA. To probe the validity of the E. coli paradigm, we examined the impact of mutations in DeltaruvAB and DeltarecU (a ruvC functional analog) on DNA repair. Under standard transformation conditions we failed to construct DeltaruvAB DeltarecG, DeltarecU DeltaruvAB, DeltarecU DeltarecG, or DeltarecU DeltarecJ strains. However, DeltaruvAB could be combined with addAB (recBCD), recF, recH, DeltarecS, DeltarecQ, and DeltarecJ mutations. The DeltaruvAB and DeltarecU mutations rendered cells extremely sensitive to DNA-damaging agents, although less sensitive than a DeltarecA strain. When damaged cells were analyzed, we found that RecU was recruited to defined double-stranded DNA breaks (DSBs) and colocalized with RecN. RecU localized to these centers at a later time point during DSB repair, and formation was dependent on RuvAB. In addition, expression of RecU in an E. coli ruvC mutant restored full resistance to UV light only when the ruvAB genes were present. The results demonstrate that, as with E. coli RuvABC, RuvAB targets RecU to recombination intermediates and that all three proteins are required for repair of DSBs arising from lesions in chromosomal DNA.

Bacillus subtilis↗
PubMed2006

Functions of multiple exonucleases are essential for cell viability, DNA repair and homologous recombination in recD mutants of Escherichia coli.

Heterotrimeric RecBCD enzyme unwinds and resects a DNA duplex containing blunt double-stranded ends and directs loading of the strand-exchange protein RecA onto the unwound 3'-ending strand, thereby initiating the majority of recombination in wild-type Escherichia coli. When the enzyme lacks its RecD subunit, the resulting RecBC enzyme, active in recD mutants, is recombination proficient although it has only helicase and RecA loading activity and is not a nuclease. However, E. coli encodes for several other exonucleases that digest double-stranded and single-stranded DNA and thus might act in consort with the RecBC enzyme to efficiently promote recombination reactions. To test this hypothesis, I inactivated multiple exonucleases (i.e., exonuclease I, exonuclease X, exonuclease VII, RecJ, and SbcCD) in recD derivatives of the wild-type and nuclease-deficient recB1067 strain and assessed the ability of the resultant mutants to maintain cell viability and to promote DNA repair and homologous recombination. A complex pattern of overlapping and sometimes competing activities of multiple exonucleases in recD mutants was thus revealed. These exonucleases were shown to be essential for cell viability, DNA repair (of UV- and gamma-induced lesions), and homologous recombination (during Hfr conjugation and P1 transduction), which are dependent on the RecBC enzyme. A model for donor DNA processing in recD transconjugants and transductants was proposed.

Cell Survival↗
PubMed2006

Repair of DNA damage induced by bile salts in Salmonella enterica.

Exposure of Salmonella enterica to sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium glycocholate, sodium taurocholate, or sodium glycochenodeoxycholate induces the SOS response, indicating that the DNA-damaging activity of bile resides in bile salts. Bile increases the frequency of GC --> AT transitions and induces the expression of genes belonging to the OxyR and SoxRS regulons, suggesting that bile salts may cause oxidative DNA damage. S. enterica mutants lacking both exonuclease III (XthA) and endonuclease IV (Nfo) are bile sensitive, indicating that S. enterica requires base excision repair (BER) to overcome DNA damage caused by bile salts. Bile resistance also requires DinB polymerase, suggesting the need of SOS-associated translesion DNA synthesis. Certain recombination functions are also required for bile resistance, and a key factor is the RecBCD enzyme. The extreme bile sensitivity of RecB-, RecC-, and RecA- RecD- mutants provides evidence that bile-induced damage may impair DNA replication.

Amino Acid Substitution↗
PubMed2006

Roles of PriA protein and double-strand DNA break repair functions in UV-induced restriction alleviation in Escherichia coli.

It has been widely considered that DNA modification protects the chromosome of bacteria E. coli K-12 against their own restriction-modification systems. Chromosomal DNA is protected from degradation by methylation of target sequences. However, when unmethylated target sequences are generated in the host chromosome, the endonuclease activity of the EcoKI restriction-modification enzyme is inactivated by the ClpXP protease and DNA is protected. This process is known as restriction alleviation (RA) and it can be induced by UV irradiation (UV-induced RA). It has been proposed that chromosomal unmethylated target sequences, a signal for the cell to protect its own DNA, can be generated by homologous recombination during the repair of damaged DNA. In this study, we wanted to further investigate the genetic requirements for recombination proteins involved in the generation of unmethylated target sequences. For this purpose, we monitored the alleviation of EcoKI restriction by measuring the survival of unmodified lambda in UV-irradiated cells. Our genetic analysis showed that UV-induced RA is dependent on the excision repair protein UvrA, the RecA-loading activity of the RecBCD enzyme, and the primosome assembly activity of the PriA helicase and is partially dependent on RecFOR proteins. On the basis of our results, we propose that unmethylated target sequences are generated at the D-loop by the strand exchange of two hemi-methylated duplex DNAs and subsequent initiation of DNA replication.

Chromosomes, Bacterial↗
PubMed2000

Identification of a DNA-binding domain and an active-site residue of pseudorabies virus DNase.

The pseudorabies virus (PRV) DNase gene has an open reading frame of 1476 nt, capable of coding a 492-residue protein. A previous study showed that PRV DNase is an alkaline exonuclease and endonuclease, exhibiting an Escherichia coli RecBCD-like catalytic function. To analyse its catalytic mechanism further, we constructed a set of clones truncated at the N-terminus or C-terminus of PRV DNase. The deleted mutants were expressed in E. coli with the use of pET expression vectors, then purified to homogeneity. Our results indicate that (1) the region spanning residues 274-492 exhibits a DNA-binding ability 7-fold that of the intact DNase; (2) the N-terminal 62 residues and the C-terminal 39 residues have important roles in 3'-exonuclease activity, and (3) residues 63-453 are responsible for 5'- and 3'-exonuclease activities. Further chemical modification of PRV DNase revealed that the inactivation of DNase by diethyl pyrocarbonate, which was reversible on treatment with hydroxylamine, seemed to be attributable solely to the modification of histidyl residues. Because the herpesviral DNases contained only one well-conserved histidine residue, site-directed mutagenesis was performed to replace His(371) with Ala. The mutant lost most of its nuclease activity; however, it still exhibited a wild-type level of DNA-binding ability. In summary, these results indicate that PRV DNase contains an independent DNA-binding domain and that His(371) is the active-site residue that has an essential role in PRV DNase activity.

Amino Acid Sequence↗
PubMed2000

[Homologous recombination between direct repeats of chromosomal segments comprising heterozygotic duplications in Escherichia coli].

In conjugational matings between double mutants for the deo operon of Escherichia coli, haploid recombinants and extended tandem duplications deoC deoD/deoA deoB::Tn5 with the DeoC+DeoA+DeoB+DeoD- phenotype are formed (the deoD+ allele is not expressed due to the polar effect of the Tn5 insertion). Selection for the expression of the recessive deoC deoD alleles (in the thyA genome) leads to the segregation of haploid clones by duplications and also of clones that retain the diploidy but that are homozygous for deoC deoD. In addition to haploids, diploid clones retaining the duplications have also been found among the DeoD+ segregants. The phenotype of segregants retaining the duplication shows that they were formed by an unequal exchange between sister chromosomes. A comparison of segregation frequency of haploid and diploid DeoD+ clones in rec+ and recBC sbcB sbcC strains shows that duplications in the rec+ genome are more stable. On this basis, it is assumed that the RecBCD pathway possibly makes a greater contribution than the RecF pathway to the preservation of heterozygous duplications playing an important role in the evolution of prokaryotes.

Chromosomes, Bacterial↗
PubMed2001

[Homologous recombination and chromosomal rearrangements in Escherichia coli strains carrying a heterozygous tandem duplication].

Heterozygous tandem duplications formed in conjugational matings in Escherichia coli provides a convenient model system for studying the evolution of bacterial chromosome. Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes. In this work, an extended tandem duplication in the deo operon of E. coli carrying deoA deoB::Tn5/deoC deoD thr::Tn9 alleles was examined. Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3 mutations. The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ (or recF) genotype. It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of "adaptive" recombination.

Alleles↗
PubMed2002

[Effect of mutations for the ruvABC genes on recombination between direct DNA repairs in Escherichia coli strains carrying extended tandem duplication].

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants for ruvABC genes. Homologous recombination in duplications of rec+ strains and in recBC sbcB, recQ and recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.

Adenosine Triphosphatases↗
PubMed2005

[Unequal genetic exchange in Escherichia coli tandem duplications may represent a special pathway of homologous recombination].

Heterozygous tandem duplications that appear in Escherichia coli conjugation matings segregate different types of haploid and diploid recombinants because of unequal crossing over between sister chromosomes. As shown previously, the frequency of segregants in the extended duplication D104 (approximately 150 kb or more than 3 min of the genetic map) heterozygous for E. coli deo-operon genes (deoA deoB::Tn5/deoC deoD) is not decreased in strains with defective RecBCD and RecF recombination pathways. Analysis of a shorter duplication of this type (approximately 46 kb) showed that the frequency of segregants in the strain recBC sbcBC recF was similar to that in a strain with undamaged system of recombination. Thus, genetic exchange between direct DNA repeats in tandem duplications may follow a special pathway of homologous recombination, which is independent of the recBC and recF genes.

Crossing Over, Genetic↗
PubMed2002

[Some genetic features of adaptive mutation].

The research based on the Escherichia coli FC40 showed that adaptive mutations required the enzymes of RecBCD recombination pathway and some unknown proteins of SOS response, and the mutation spectrum of lac+ revertants is single-base deletions in the small mononucleotide repeats. Some evidence showed that the revertants with adaptive mutations partly come from one (or some) subset of transient hypermutable subpopulation of cells, in which high frequently losing of transposons and genome-wide mutations were observed. It was suggested that this kind of transient hypermutability may be due to the transient deficient activity of mismatch repair (MMR) system, or a defective epsilon unit of DNA polymerase III generated by mistranslation. Although other systems demonstrated some different mechanisms from FC40, all research works suggested that, adaptive mutations occurred in nondividing or nongrowing cells under environmental stresses, for example, starvation, displayed different genetic features from growth-dependent spontaneous mutation.

English Abstract↗
PubMed1991

Inhibition of recA-mediated strand exchange by adducts of azacytosine-containing DNA and the EcoRII methylase.

Wild type Escherichia coli cells containing elevated levels of DNA (cytosine-5)methyltransferases have increased sensitivity to the toxic effects of 5-azacytidine. The methyltransferases form tight binding complexes with azacytosine in DNA which could interfere with the recA recBCD repair pathway which is largely responsible for cell survival after treatment with the drug. We therefore determined if these complexes interfered with recA-mediated strand exchange in vitro. 32P-Labeled DNA fragments containing a single EcoRII site, with cytosine in the (-) strand replaced by 5-azacytosine, were prepared. We investigated the effect of the EcoRII methyltransferase on recA-mediated strand exchange with homologous M13 DNA by electrophoresis in agarose gels. In the absence of the methylase the rate and extent of strand exchange of azacytosine-containing DNA is the same as control DNA. In the presence of the methyltransferase strand exchange is inhibited, but some incorporation of duplexes into recA-single-stranded DNA (ssDNA) complexes still occurs. The formation of these complexes is dependent on the length of the fragment 3' to the methylase binding site on the strand complementary to the ssDNA. The greater the length the greater the number of complexes that form. S-Adenosyl-L-methionine, which enhances binding of the methyltransferase to azacytosine-containing DNA, causes an increase in the inhibition of strand exchange and an increase in the number of inactive complexes formed. The complexes can be dissociated with guanidinium chloride which denatures the methyltransferase and leads to release of the (+) strand. The (-) strand remains associated with the ssDNA. This result implies that a plectonemic joint is formed between recA-ssDNA complexes and azacytosine-containing DNA-methyltransferase complexes. However, branch migration in these complexes is inhibited. Denaturation of the methyltransferase allows branch migration to proceed to completion, releasing the (+) strand.

Cytosine↗
PubMed1990

[Frequency of recombination exchanges in Escherichia coli K-12: genetic determinants controlling this frequency].

RecF, recQ, ruv, recJ and recN genes of so called RecF pathway of recombination appear to be not silent on the RecBCD pathway also. These genes are responsible for the frequency of recombination exchanges per unit length of DNA. The list: recF::Kmr greater than recQ::Tn3 greater than ruv54 greater than recJ::Tn9 demonstrated the efficiency of inhibition of recombination exchanges by these mutations. The recN262 mutation gives a feeble contrary effect. It slightly increases the frequency of recombination exchanges.

Escherichia coli↗
PubMed1993

[Increased frequency of unequal crossing over in recBC mutant cells in conjugational crosses in Escherichia coli K-12].

The formation of heterozygous tandem duplications was studied in conjugational crosses of Escherichia coli using mutations for deo-operon genes. The frequency of tandem duplications in recF::Tn3 and recBC sbcB mutants was not substantially changed from that of the wild type strain. In contrast, the frequency of duplications was increased about ten-fold when using recBC (sbcB+) mutants as recipients. This result can be explained by a participation of the RecBCD protein in the pairing of chromosomes during recombination. We also found that HfrH thyA recBC (sbcB+) bacteria grow very poorly on rich medium and that good growth is restored by conversion from the Hfr to the F+ state, as well as by mutations suppressing the RecBC phenotype; such F+ strains retain sensitivity to UV light and to mitomycin C, which are characteristic of the recBC phenotype.

Conjugation, Genetic↗
PubMed1996

[Attenuation of type I restriction in Escherichia coli: effect of the ard gene in UV-irradiated cells].

The effect of conjugative plasmids ColIb-P9 (incI1) and pKM 101 (incN), containing the active ard gene, on the efficiency of EcoK restriction of nonmodified phage lambda .0 in UV-irradiated Escherichia coli cells was studied. ard-Dependent antirestriction enzyme activity was shown to decrease in UV-irradiated cells. The efficiency of action of the ard plasmid gene on lambda .0 was also shown not to depend on cell helicases RecBCD and UvrD, in contrast to the UV-induced alleviation of EcoK restriction (SOS alleviation).

Deoxyribonucleases, Type I Site-Specific↗