Identification of the recA gene of Streptococcus pneumoniae.
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Amino Acid Sequence↗
Biomedical subjects
R Devoret
Publications and source records attributed to R Devoret.
A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments.
The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein. If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction. In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction. Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein. The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step. In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype. Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities. RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody. However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI. RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis. These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments.
Adenosine Triphosphatases↗
PsiB, and anti-SOS protein, is transiently expressed by the F sex factor during its transmission to an Escherichia coli K-12 recipient.
PsiB, an anti-SOS protein, shown previously to prevent activation of RecA protein, was purified from the crude extract of PsiB overproducing cells. PsiB is probably a tetrameric protein, whose subunit has a sequence-deduced molecular mass of 15741 daltons. Using an immuno-assay with anti-PsiB antibodies, we have monitored PsiB cell concentrations produced by F and R6-5 plasmids: the latter type produces a detectable level of PsiB protein while the former does not. The discrepancy can be assigned to a Tn10 out-going promoter located upstream of psiB. When we inserted a Tn10 promoter upstream of F psiB, the F PsiB protein concentration reached the level of R6-5 PsiB. We describe here the physiological role that PsiB protein may have in the cell and how it causes an anti-SOS function. We observed that PsiB protein was transiently expressed by a wild-type F sex factor during its transmission to an Escherichia coli K-12 recipient. In an F+ x F- cross, PsiB concentration increased at least 10-fold in F- recipient bacteria after 90 minutes and declined thereafter; the psiB gene may be repressed when F plasmid replicates vegetatively. PsiB protein may be induced zygotically so as to protect F single-stranded DNA transferred upon conjugation. PsiB protein, when overproduced, may interfere with RecA protein at chromosomal single-stranded DNA sites generated by discontinuous DNA replication, thus causing an SOS inhibitory phenotype.
Bacterial Proteins↗
Identification and expression of the cDNA of KIN17, a zinc-finger gene located on mouse chromosome 2, encoding a new DNA-binding protein.
We report the cloning of KIN17 cDNA, 1414 bp long with an ORF of 391 residues showing a zinc finger and nuclear localization signals. By recloning the cDNA into an appropriate vector, we produced kin17 protein in E. coli, purified it partially and shown that kin17 protein binds to double-stranded DNA. The KIN17 gene was localized by cytogenetic mapping in mouse chromosome 2, band A. Genomic sequences homologous to KIN17 cDNA were detected also in rat and human DNAs. KIN17 mRNA is highly expressed in rodent transformed AtT-20 neuroendocrine cells whereas it can be detected only in the total RNA of mouse embryos and various normal adult tissues by reverse transcription and PCR amplification. The mouse nuclear kin17 protein was identified by a local small structural similarity with E.coli recA protein. Kin17 and recA have only 39 amino acid residues in a region that might be involved in DNA-binding.
Amino Acid Sequence↗
Substitution of UmuD' for UmuD does not affect SOS mutagenesis.
In order to study the role of UmuDC proteins in SOS mutagenesis, we have constructed new Escherichia coli K-12 strains to avoid i) over-production of Umu proteins, ii) the formation of unwanted mixed plasmid and chromosomal Umu proteins upon complementation. We inserted a mini-kan transposon into the umuD gene carried on a plasmid. The insertion at codon 24 ends protein translation and has a polar effect on the expression of the downstream umuC gene. We transferred umuD24 mutation to the E coli chromosome. In parallel, we subcloned umuD+ umuC+ or umuD' umuC+ genes into pSC101, a low copy number plasmid. In a host with the chromosomal umuD24 mutation, plasmids umuD+ umuC+ or umuD' umuC+ produced elevated resistance to UV light and increased SOS mutagenesis related to a gene dosage of about 3. UV mutagenesis was as high in umuD' umuC+ hosts devoid of UmuD+ protein as in umuD+ umuC+ hosts. UmuD' protein, the maturated form of UmuD, can substitute for UmuD in SOS mutagenesis.
Bacterial Proteins↗
A RecA protein mutant deficient in its interaction with the UmuDC complex.
recA1730 is a dominant point mutation preventing SOS mutagenesis. We demonstrate here that: i) RecA1730 fails to produce mutagenesis even though UmuD' is formed, ii) recA1730, when complemented by recA+, can cleave LexA protein and it displays a UmuDC- phenotype in spite of adequate concentrations of matured UmuD' and UmuC proteins, iii) the Mut- phenotype caused by RecA1730 is partially alleviated by MucAB proteins, functional analogs of UmuDC. To explain the mutant phenotype, we postulate that recA1730 impairs a RecA function required for the positioning of the UmuD'C complex within the replisome at the site of lesions.
Bacterial Proteins↗
Introduction of a UV-damaged replicon into a recipient cell is not a sufficient condition to produce an SOS-inducing signal.
Three models have been proposed for the nature of the SOS-inducing signal in E. coli. One model postulates that degradation products of damaged DNA generate an SOS-inducing signal; another model surmises that the very lesions produced by UV damage constitute the SOS-inducing signal in vivo; a third model proposes that DNA damage is processed upon DNA replication to form single-stranded DNA (the SOS signal) that activates RecA protein. We tested the models by measuring SOS induction produced by introducing into recipient cells the UV-damaged DNA of 2 constructed phagemids. We used phagemids since they transferred DNA to the recipients with 100% efficiency. The origin of replication of the phagemids was either oriC from the E. coli chromosome, or oriF from F plasmid. Replication of the oriC phagemid was dependent on methylation. A UV-damaged oriC phagemid failed to induce SOS functions in a recipient cell whereas an oriF phagemid did induce them. Our results disprove the first and the second model proposed for the nature of the SOS-inducing signal. The failure of a UV-damaged oriC replicon to induce SOS can be explained by the third model if one assumes that replication of a UV-damaged oriC plasmid does not generate single-stranded DNA as does the E. coli chromosome after UV damage.
Bacteriophage lambda↗
Nucleotide sequence of the psiA (plasmid SOS inhibition) gene located on the leading region of plasmids F and R6-5.
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Amino Acid Sequence↗
The bI4 RNA mitochondrial maturase of Saccharomyces cerevisiae can stimulate intra-chromosomal recombination in Escherichia coli.
When the bI4 RNA maturase, encoded by the fourth intron of the mitochondrial cytochrome b gene of Saccharomyces cerevisiae, was expressed in Escherichia coli, formation of intra-chromosomal Lac+ recombinants was stimulated threefold. This "hyper-rec" phenotype was recA as well as recBCD dependent. The most active form of the bI4 maturase stimulated homologous recombination whereas splicing deficient mutants of bI4 maturase were either deficient in or unable to stimulate homologous recombination.
DNA Nucleotidyltransferases↗
KIN, a mammalian nuclear protein immunologically related to E. coli RecA protein.
A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa). The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus. Its level is higher in proliferating than in quiescent cells. Cell treatment with mitomycin C increases the level of the KIN protein. We sought similar proteins in other mammalian cells. Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos.
Animals↗
New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis.
To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+. With respect to other RecA functions, recA1730 was recessive to recA+. This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins.
Bacterial Proteins↗
Identification of psiB genes of plasmids F and R6-5. Molecular basis for psiB enhanced expression in plasmid R6-5.
PsiB protein of plasmid R6-5 inhibits the induction of the SOS pathway. The F sex factor also carries a psiB gene homologous to that of R6-5. Yet, it fails to inhibit SOS induction. In order to solve this difference, we characterized the psiB genes of R6-5 and F. We found that (i) the sequences of the two psiB genes share extensive homology the predicted amino acid sequences of the two proteins differing by 5 residues, (ii) the expression of R6-5 psiB is 4 times higher than F psiB gene, (iii) in plasmid R6-5, a Tn10 transposon upstream from the psiB gene enhances psiB expression. Hence, the F sex factor may be unable to prevent SOS induction for two non-exclusive reasons: (i) F PsiB protein, being slightly different from R6-5, may be less active, (ii) the level of synthesis of F PsiB protein may be insufficient to prevent SOS induction.
Amino Acid Sequence↗
PsiB polypeptide prevents activation of RecA protein in Escherichia coli.
We further characterize a novel plasmid function preventing SOS induction called Psi (Plasmid SOS Inhibition). We show that Psi function is expressed by psiB, a gene located at coordinate 54.9 of plasmid R6-5 and near oriT, the origin of conjugal transfer. Deletions and amber mutations of the psiB gene permitted us to demonstrate that PsiB polypeptide (apparent molecular weight, 12 kDa) is responsible for Psi function. PsiB protein prevents recA730-promoted mutagenesis and intra-chromosomal recombination but not recombination following conjugation. Overproduction of PsiB protein sensitizes the host cell to UV irradiation. We propose that PsiB polypeptide has an anti-SOS action by inhibiting activation of RecA protein, thus preventing the occurrence of LexA-controlled functions.
Bacterial Proteins↗
A gene encoding an SOS inhibitor is present in different conjugative plasmids.
In 9 of 20 conjugative plasmids of different incompatibility groups, including F and R100 (or R6-5), coexist two sequences which are homologous, respectively, to the gene psiB, which encodes an inhibitor of SOS induction, and to the gene ssb, which encodes a single-stranded-DNA-binding protein.
Bacterial Proteins↗
An inhibitor of SOS induction, specified by a plasmid locus in Escherichia coli.
Plasmid R6-5 contains a locus whose product inhibits induction of sfiA and prophage lambda in a recA441 mutant at 42 degrees C and in a recA+ host after treatment with nalidixic acid. This plasmidic SOS-inhibition locus (psi) is situated on an 8.1-kilobase DNA fragment near oriT, the origin of plasmid R6-5 conjugational transfer. Loss of the Psi function, resulting from the insertion of Tn3 into psi+, greatly reduced the synthesis of two proteins, designated PsiA (Mr 24,500) and PsiB (Mr 12,500). Using host cells in which there was an inactive LexA repressor, we found that Psi function does not act by interfering with the expression of the SOS pathway. The Psi function may affect the generation of an SOS signal. We postulate that during the course of evolution, the Psi function has been selected in some conjugative plasmids so as to permit them to transfer single-stranded DNA without generating an SOS signal.
Biological Evolution↗
SOS induction by thermosensitive replication mutants of miniF plasmid.
MiniF, a 9.3 kb fragment of the dispensable F plasmid, carries genes necessary for its replication and partition as well as for the expression of an SOS signal. The arrest of replication of a thermo-sensitive miniFts at 42 degrees C induced SOS functions such as prophage lambda, sfiA expression, W-reactivation of UV-irradiated phage lambda. Two miniF ts9 and ts17 mutations were located within the KpnI fragment (43.6-46.9) in the minimal oriS replicon. Blocking miniF replication by incBC+ incompatibility genes situated in trans on a second plasmid also induced SOS functions. In contrast, if miniFts17 plasmid escaped the replication block at 42 degrees C by being inserted into pR325, there was no SOS induction. SOS induction by the arrest of miniF replication required the miniF lynA+ locus in cis, the host recA+ and lexA+ genes. We found that SOS induction was increased greatly near the stationary phase and that cell viability declined. During host cell exponential growth, miniFts9 and miniFts17 plasmids were lost rapidly, although SOS induction persisted for several cell generations. We postulate that lynA expresses a persistent product that may lead to the unwinding of chromosomal DNA.
Bacterial Proteins↗
A yeast protein analogous to Escherichia coli RecA protein whose cellular level is enhanced after UV irradiation.
In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K 12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD+) strains carrying mitochondrial DNA or not (rho0). This was also true for the following DNA-repair deficient (rad-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-delta, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.
Antibodies↗
Mini-F plasmid-induced SOS signal in Escherichia coli is RecBC dependent.
Dispensable replicons such as F plasmid [95 kilobases (kb)] or its mini-derivatives such as mini-F (9.3 kb) or lambda mini-F efficiently induced cellular SOS genes such as sfiA (sulA) when they were damaged by UV irradiation and then introduced into a recipient bacterium. To generate an SOS signal, UV light-damaged mini-F or mini-F conditional mutants deficient in replication required that the bacterial RecBC enzyme retained some activity different from the nuclease activity that was dispensable. In contrast, UV light-damaged F plasmid produced an SOS signal independently of the activity of the RecBC enzyme and of the expression of the mini-F, -H, and -G proteins. Our findings are consistent with a picture in which the SOS signal is constituted by stretches of single-stranded DNA on a replicon. Moreover, our present data combined with other data previously published lead to the hypothesis that the SOS signal induced by mini-F plasmid is located in trans on the host chromosome, whereas the one generated by UV light-damaged F plasmid is in cis on the transferred DNA.
Chromosomes, Bacterial↗