Impact of DNA gyrase inhibition by antisense ribozymes on rec A in <Emphasis Type="Italic">E. coli</Emphasis>

The chromosome of E. coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. Regulation of DNA supercoiling yields a complex spectrum of effects on the E. coli recA system. Previous studies indicated that inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the recA system. Here we show that antisense ribozymes that act on the DNA gyrase A subunit can also induce recA. We used real time PCR and immunoblot to analyze the impact of DNA gyrase A inhibition by antisense ribozymes on recA expression. When gyrase A was inhibited by the RNase P mediated antisense ribozymes the expression of recA was induced around 130-fold as seen by real time PCR analysis. This suggests that repair pathway is induced by antisense ribozymes against DNA gyrase A and the damage produced by these ribozymes may be similar to that produced by fluroquinolones.     

An<Emphasis Type="Italic"> Arabidopsis</Emphasis> homologue of bacterial RecA that complements an<Emphasis Type="Italic"> E. coli recA</Emphasis> deletion is targeted to plant mitochondria

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mt recA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mt recA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mt recA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mt recA in homologous recombination and/or repair in Arabidopsis mitochondria.     

Molecular characterization of regulatory elements controlling expression of the Bacillus subtilis recA+ gene.

Expression of the Bacillus subtilis recA gene is induced following DNA damage as well as during the development of the competent state. DNA damage-induction of the recA gene occurs by a RecA-dependent mechanism, whereas competence-induction occurs by a RecA-independent mechanism. To examine the molecular mechanisms that control the expression of the recA gene, a deletion analysis of the recA promoter region was performed. A regulatory region that is required for repression of recA expression was identified upstream of the recA promoter. Deletion of this regulatory region derepressed expression and abolished damage-induction of the recA promoter. Within this region are sequences similar to the consensus sequence that has been identified within DNA damage-inducible promoter regions of other B subtilis genes. Another regulatory region was identified that is required for the RecA-independent, competence-specific induction of the recA gene. Deletion of these sequences significantly reduced competence-induction of the recA promoter.     

Identification, isolation and sequencing of the recA gene of Streptomyces lividans TK24

Abstract An internal fragment of the recA gene of Streptomyces cattleya was amplified by the polymerase chain reaction (PCR) employing degenerate oligonucleotide primers. Using this fragment as a hybridization probe, a recA homologous gene could be shown in each tested Streptomyces strain. A 4.4 kb Bam HI fragment which carried the complete recA gene was isolated from Streptomyces lividans TK24. Sequence analysis suggested that the coding region of the recA gene consists of 1122 bp. The highest similarity (∼78%) could be detected to the recA genes of Mycobacterium tuberculosis and Mycobacterium leprae . After fusion with an E. coli promoter the S. lividans recA gene could partially complement an Escherichia coli recA mutant.     

Molecular cloning and expression in Escherichia coli of the recA gene of Legionella pneumophila

Interspecific complementation of an Escherichia coli recA mutant with a Legionella pneumophila genomic library was used to identify a recombinant plasmid encoding the L. pneumophila recA gene. Recombinant E. coli strains harbouring the L. pneumophila recA gene were isolated by replica-plating bacterial colonies on medium containing methyl methanesulphonate (MMS). MMS-resistant clones were identified as encoding the L. pneumophila recA analogue by their ability to protect E. coli HB101 from UV exposure and promote homologous recombination. Subcloning of selected restriction fragments and Tn5 mutagenesis localized the recA gene to a 1.7 kb Bg/II-EcoRI fragment. Analysis of minicell preparations harbouring a 1.9 kb EcoRI fragment containing the recA coding segment revealed a single 37.5 kDa protein. Insertional inactivation of the cloned recA gene by Tn5 resulted in the disappearance of the 37.5 kDa protein, concomitant with the loss of RecA function. The L. pneumophila recA gene product did not promote induction of a lambda lysogen; instead, the presence of the heterologous recA gene caused a significant reduction in spontaneous and mitomycin-C-induced prophage induction in recA+ and recA E. coli backgrounds. Despite the lack of significant genetic homology between the L. pneumophila recA gene and the E. coli counterpart, the L. pneumophila RecA protein was nearly identical to that of E. coli in molecular mass, and the two proteins showed antigenic cross-reactivity. Western blot analysis of UV-treated L. pneumophila revealed a significant increase in RecA antigen in irradiated versus control cells, suggesting that the L. pneumophila recA gene is regulated in a manner similar to that of E. coli recA.     

Functional characteristics of the recA gene from Serratia marcescens strain Sb

The cloned recA gene from Serratia marcescens Sb was expressed and complemented defects in the UV repair, recombination, and SOS induction of an Escherichia coli host deleted for recA. Moreover, the Serratia gene, recA (Sm), supported the same frequency of recombination per unit length of DNA as did the homologous Escherichia coli gene, recA(Ec).     

Interaction of the recA protein of Escherichia coli with single-stranded DNA

The interaction of recA protein with single-stranded (ss) phi X174 DNA has been examined by means of a nuclease protection assay. The stoichiometry of protection was found to be 1 recA monomer/approximately 4 nucleotides of ssDNA both in the absence of a nucleotide cofactor and in the presence of ATP. In contrast, in the presence of adenosine 5'-O-(thiotriphosphate) (ATP gamma S) the stoichiometry was 1 recA monomer/approximately 8 nucleotides. No protection was seen with ADP. In the absence of a nucleotide cofactor, the binding of recA protein to ssDNA was quite stable as judged by equilibration with a challenge DNA (t1/2 approximately 30 min). Addition of ATP stimulated this transfer (t1/2 approximately 3 min) as did ADP (t1/2 approximately 0.2 min). ATP gamma S greatly reduced the rate of equilibration (t1/2 greater than 12 h). Direct visualization of recA X ssDNA complexes at subsaturating recA protein concentrations using electron microscopy revealed individual ssDNA molecules partially covered with recA protein which were converted to highly condensed networks upon addition of ATP gamma S. These results have led to a general model for the interaction of recA protein with ssDNA.     

Cloning of the recA gene of Bordetella pertussis and characterization of its product.

A recA gene of Bordetella pertussis was identified in a plasmid library by complementation of a recA mutation in E coli and subcloned as a 2.1-kb Sph I DNA fragment. Southern hybridization experiments showed no similarity to the E coli recA gene, but very strong similarity to other Bordetella species. E coli recA mutant cells containing the B pertussis recA gene at high gene dosage were resistant to DNA-damaging agents such as methyl methane sulfonate or 4-nitroquinoline-N-oxide, displayed induction of SOS functions, and were able to promote DNA recombination, but not induction of phage lambda. The latter phenotype distinguishes the B pertussis recA gene product from the corresponding proteins from most other Gram-negative organisms. Amino acid sequence comparisons revealed a high degree of structural conservation between prokaryotic RecA proteins.     

On the polymerization state of recA in the absence of DNA

We present an electron microscopy study on the polymerization state of recA in the absence of DNA. In solution recA exists as monomers, small complexes not clearly longer than wide (approximately 9-18 nm), and filaments (diameter approximately 11 nm and variable lengths). We have attempted to quantify the relative amounts of these species by length measurements of the particles on electron micrographs. The percentages of each of these types was found to depend on recA concentration, temperature and presence in the incubation mixture of Mg2+, ATP gamma S, salt or D2O. These additives do not have an absolute effect on polymerization but rather shift the polymerization equilibrium of recA (which depends on recA concentration) up or down the concentration scale.     

Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena

In recb recC sbcB mutants genetic recombination is dependent upon the recF gene. recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background. Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background. In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages. In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101. These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.     

A loss of uvrA function decreases the induction of the SOS functions recA and umuC by mitomycin C in Escherichia coli

We have studied the levels of recA and umuC protein synthesis in Escherichia coli as a probe for regulatory and mechanistic events involved in mitomycin C mutagenesis. Both RecA and UmuC protein induction were greatly stimulated by mitomycin C in the wild-type strain, reached a peak at about 60 min for the recA gene, and at 90 min for the umuC gene, respectively, and maintained a plateau. The induction was blocked by recA and lexA(Ind-) mutations that conferred no mutagenesis on the cell. Mutation affecting uvrA protein markedly decreased induction of the recA gene as well as the umuC gene by mitomycin C. The results established that UvrA protein is involved in the induction of recA and umuC, and account, at least in part, for the mitomycin C nonmutability of uvrA mutants.     

Biochemical properties of the Escherichia coli recA430 protein. Analysis of a mutation that affects the interaction of the ATP-recA protein complex with single-stranded DNA

The biochemical properties of the recA430 protein have been examined and compared to those of wild-type recA protein. We find that, while the recA430 protein possesses ssDNA-dependent rATP activity, this activity is inhibited by the Escherichia coli single-stranded DNA binding protein (SSB protein) under many conditions that enhance wild-type recA protein rATPase hydrolysis. Stimulation of rATPase activity by SSB protein is observed only at high concentrations of both rATP (greater than 1 mM) and recA430 protein (greater than 5 microM). In contrast, stimulation of ssDNA-dependent dATPase activity by SSB protein is less sensitive to protein and nucleotide concentration. Consistent with the nucleotide hydrolysis data, recA430 protein can carry out DNA strand exchange in the presence of either rATP or dATP. However, in the presence of rATP, both the rate and the extent of DNA strand exchange by recA430 protein are greatly reduced compared to wild-type recA protein and are sensitive to recA430 protein concentration. This reduction is presumably due to the inability of recA430 protein to compete with SSB protein for ssDNA binding sites under these conditions. The cleavage of lexA repressor protein by recA430 protein is also sensitive to the nucleotide cofactor present and is completely inhibited by SSB protein when rATP is the cofactor but not when dATP is used. Finally, the steady-state affinity and the rate of association of the recA430 protein-ssDNA complex are reduced, suggesting that the mutation affects the interaction of the ATP-bound form of recA protein with ssDNA. This alteration is the likely molecular defect responsible for inhibition of recA430 protein rATP-dependent function by SSB protein. The biochemical properties observed in the presence of dATP and SSB protein, i.e. the reduced levels of both DNA strand exchange activity and cleavage of lexA repressor protein, are consistent with the phenotypic behavior of recA430 mutations.     

New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity.

The RecA protein plays a key role in Escherichia coli recombination and DNA repair. We have created new recA mutants with mutations in the vicinity of the recA430 mutation (Gly-204----Ser) which is known to affect RecA coprotease activity. Mutants carrying recA659 or recA611, located 3 and 7 amino acids downstream of residue 204, respectively, lose all RecA activities, while the mutant carrying recA616, which is located at 12 amino acids from this residue, keeps the coprotease activity but is unable to promote recombination. Complementation experiments show that both mutations recA611 and recA659 are dominant over the wild-type or recA430 allele while recA616 seems to be recessive to recA+ and dominant over recA430. It is suggested that these mutations are located in RecA domains which direct conformational modifications.     

Evidence that the phr+ gene enhances the ultraviolet resistance of Escherichia coli recA strains in the dark

An Escherichia coli recA phr+ purA strain was more resistant to ultraviolet radiation than its isogenic derivative recA phr+ purA+ in the absence of photoreactivating light, whereas their nearly isogenic derivative recA phr showed most UV-induced lethality. The amounts of photoreactivating enzyme (PRE) per cell in the recA phr+ purA was higher than in the recA phr+ purA+. The recA phr is defective for photoreactivation. Thus, in the recA strain, UV resistance in the dark increased in proportion to the amounts of PRE per cell, suggesting that PRE participates in the process of dark repair of UV-damaged DNA.     

Increase of the DNA strand assimilation activity of recA protein by removal of the C terminus and structure-function studies of the resulting protein fragment

A proteolytic fragment of recA protein, missing about 15% of the protein at the C terminus, was found to promote assimilation of homologous single-stranded DNA into duplex DNA more efficiently than intact recA protein. This difference was not found if Escherichia coli single-stranded DNA binding protein was present. The ATPase activity of both intact recA protein and the fragment was identical. The difference in strand assimilation activity cannot be due to differences in single-stranded DNA affinity, since both the fragment and intact proteins bind to single-stranded DNA with nearly identical affinities. However, the fragment was found to bind double-stranded DNA more tightly and to aggregate more extensively than recA protein; both of these properties may be important in strand assimilation. Aggregation of the fragment was extensive in the presence of duplex DNA under the same condition where recA protein did not aggregate. The double-stranded DNA binding of both recA protein and the fragment responds to nucleotide cofactors in the same manner as single-stranded DNA binding, i.e. ADP weakens and ATP gamma S strengthens the association. The missing C-terminal region of recA protein includes a very acidic region that is homologous to other single-stranded DNA binding proteins and which has been implicated in DNA binding modulation. This C-terminal region may serve a similar function in recA protein, possibly inhibiting double-stranded DNA invasion. The possible role of the enhanced double-stranded DNA affinity of the fragment protein in the mechanism of strand assimilation is discussed.     

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.     

Functional domains of Escherichia coli recA protein deduced from the mutational sites in the gene

Summary The sites of recA mutations of Escherichia coli, recA441 (tif-1), recA1, recA430 (lexB30) and recA44, were determined by analyses of the nucleotide sequences. All mutations are single point missense mutations within the coding region of the recA gene. In the recA441, recA1, recA430 and recA44 proteins, the 38th, 160th, 204th, and 246th amino acids, respectively, from the amino terminal ends are altered. Based on the properties of these mutant proteins and modified forms of recA protein, the locations of various regions of the recA protein that are involved in binding with ATP, binding with single-stranded DNA, hydrolysis of ATP, interaction between the recA protein molecules and interaction with the cI or lexA repressors are mapped on the primary structure of the protein.     

Cloning and preliminary characterization of srf-2020 and srf-801, the recF partial suppressor mutations which map in recA of Escherichia coli K-12

We have located the single nucleotide changes suffered in recA sequence of 2 recF partial suppressor mutations: srf-2020 at codon 121 and srf-801 at codon 257. srf-2020 changes codon 121 from threonine (ACC) to isoleucine (ATC). srf-801 changes codon 257 from glutamine (CAG) to proline (CCG). Consequently these mutations were renamed recA2020 and recA801 respectively. Preliminary characterization of recA2020 revealed that it is transdominant to recA+, like recA803, another recF partial suppressor. Interactions of recA2020 with recA803 were examined using genetic studies. Heterozygotes containing recA2020 and recA803 failed to produce a synergistic suppression effect in suppressing the recF deficiency. Presence of both recA2020 and recA803 mutations in the same recA gene also failed to produce any greater amount of UV resistance to a uvrA6recF143del(recA) strain indicating no interactions between these suppressors.     

Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant.

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.