Exchange of recA protein between adjacent recA protein-single-stranded DNA complexes

We have examined the exchange of recA protein between stable complexes formed with single-stranded DNA (ssDNA) and (a) other complexes and (b) a pool of free recA protein. We have also examined the relationship of ATP hydrolysis to these exchange reactions. Exchange was observed between two different recA X ssDNA complexes in the presence of ATP. Complete equilibration between two sets of complexes occurred with a t1/2 of 3-7 min under a set of conditions previously found to be optimal for recA protein-promoted DNA strand exchange. Approximately 200 ATPs were hydrolyzed for every detected migration of a recA monomer from one complex to another. This exchange occurred primarily between adjacent complexes, however. Little or no exchange was observed between recA X ssDNA complexes and the free recA protein pool, even after several hundred molecules of ATP had been hydrolyzed for every recA monomer present. ATP hydrolysis is not coupled to complete dissociation or association of recA protein from or with recA X ssDNA complexes under these conditions.     

recA protein-promoted ATP hydrolysis occurs throughout recA nucleoprotein filaments

When recA protein binds cooperatively to single-stranded DNA to form filamentous nucleoprotein complexes, it becomes competent to hydrolyze ATP. No correlation exists between the ends of such complexes and the rate of ATP hydrolysis. ATP hydrolysis is not, therefore, restricted to the terminal subunits on cooperatively bound recA oligomers, but occurs throughout the complex. Similarly, during recA protein-promoted branch migration (during DNA strand exchange), ATP hydrolysis is not restricted to recA protein monomers at the branch point. DNA cofactors of lengths varying from 16 bases to over 12,000 bases support ATP hydrolysis. The maximum value of kcat at infinite DNA concentration is about 29/min independent of the length of the DNA cofactor. The apparent dissociation constant, however, is a strong function of DNA length, providing evidence for a minimum site size of 30-50 bases for efficient binding of recA protein.     

Negative complementation of recA protein by recA1 polypeptide: in vivo recombination requires a multimeric form of recA protein

Summary Recombination in vivo was studied in recA ^- heterozygous lacZ merodiploids by performing -galactosidase assays after infection with precA ⁺. Recombination as measured by -galactosidase production was a linear function of pecA ⁺ multiplicity of infection (MOI) when the strain contained a deletion of the chromosomal recA gene. However, when the strain carried a recA1 missense allele, a higher precA ⁺ MOI was required to obtain levels of recombination comparable to the (recA) strain, and the slope of the dose-response curve increased to approximately two. It is proposed that negative complementation occurs in mixed tetramers of wild-type and missense recA polypeptides, and that in vivo recombination is a property of a multimeric form of recA protein.     

Gene sequence of recA + and construction of recA mutants of Vibrio cholerae

The recA ⁺ gene of Vibrio cholerae O1 has been cloned, its nucleotide sequence determined and the product characterized. A deletion mutation was constructed in the recA gene and mutants showed the typical sensitivity to UV and to DNA-damaging agents, as well as an inability to mediate homologous DNA recombination. The chromosomal recA deletion mutants in V. cholerae do not show altered virulence in the infant mouse cholera model and are thus ideal strains for use in complementation studies.     

Roles of recA mutant allele (recA495) in frameshift mutagenesis

The chemical carcinogen N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) induces frameshift mutations located within two types of specific sequences (mutation hot spots): i) contiguous guanine sequences and ii) alternating GC sequences. The genetic requirements of these frameshift events were investigated using specific reversion assays. AAF-induced -2 frameshift mutagenesis at alternating GC sequences is peculiar in that it requires a LexA- controlled function which is not UmuDC and occurs in the absence of RecA protein, provided the SOS regulon is derepressed. Moreover, the non-activated form of the RecA protein was shown to act as an inhibitor in this mutation pathway. As we were interested in elucidating this mutation pathway, we have developed a convenient spot reversion assay specific for the detection of this class of mutations. This assay allowed us to isolate E coli mutants affected either in repair or mutagenesis functions. One particular mutant, recA495, is very sensitive to UV and N-AcO-AAF, and is defective in recombination and UV mutagenesis. The RecA495 protein exhibits very low binding to both single- and double-stranded DNA. We show that when the SOS regulon is derepressed, the recA495 allele has two contrasting roles in frameshift mutagenesis: i) it prevents the induction of -1 frameshift mutations at repetitive sequences and ii) it is permissive for the induction of -2 frameshift mutations within alternating GC sequences.     

Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins.

The recA730 mutation results in constitutive SOS and prophage induction. We examined biochemical properties of recA730 protein in an effort to explain the constitutive activity observed in recA730 strains. We find that recA730 protein is more proficient than the wild-type recA protein in the competition with single-stranded DNA binding protein (SSB protein) for single-stranded DNA (ssDNA) binding sites. Because an increased aptitude in the competition with SSB protein has been previously reported for recA441 protein and recA803 protein, we directly compared their in vitro activities with those of recA730 protein. At low magnesium ion concentration, both ATP hydrolysis and lexA protein cleavage experiments demonstrate that these recA proteins displace SSB protein from ssDNA in a manner consistent with their in vivo repressor cleavage activity, i.e. recA730 protein > recA441 protein > recA803 protein > recAwt protein. Additionally, a correlation exists between the proficiency of the recA proteins in SSB protein displacement and their rate of association with ssDNA. We propose that an increased rate of association with ssDNA allows recA730 protein to displace SSB protein from the ssDNA that occurs naturally in Escherichia coli and thereby to become activated for the repressor cleavage that leads to SOS induction. RecA441 protein is similarly activated for repressor cleavage; however, in this case, significant SSB protein displacement occurs only at elevated temperature. At physiological magnesium ion concentration, we argue that recA803 protein and wild-type recA protein do not displace sufficient SSB protein from ssDNA to constitutively induce the SOS response.     

Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele

Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264→Glu) and recA665 (Tyr264→His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.     

Cloning of the recA gene of Neisseria gonorrhoeae and construction of gonococcal recA mutants.

Interspecific complementation of an Escherichia coli recA mutant was used to identify recombinant plasmids within a genomic cosmid library derived from Neisseria gonorrhoeae that carry the gonococcal recA gene. These plasmids complement the E. coli recA mutation in both homologous recombination functions and resistance to DNA damaging agents. Subcloning, deletion mapping, and transposon Tn5 mutagenesis were used to localize the gonococcal gene responsible for suppression of the E. coli RecA- phenotype. Defined mutations in and near the cloned gonococcal recA gene were constructed in vitro and concurrently associated with a selectable genetic marker for N. gonorrhoeae and the mutated alleles were then reintroduced into the gonococcal chromosome by transformation-mediated marker rescue. This work resulted in the construction of two isogenic strains of N. gonorrhoeae, one of which expresses a reduced proficiency in homologous recombination activity and DNA repair function while the other displays an absolute deficiency in these capacities. These gonococcal mutants behaved similarly to recA mutants of other procaryotic species and displayed phenotypes consistent with the data obtained by heterospecific complementation in an E. coli recA host. The functional activities of the recA gene products of N. gonorrhoeae and E. coli appear to be highly conserved.     

Construction of a recA mutant of Azospirillum lipoferum and involvement of recA in phase variation

The plant-growth promoting rhizobacterium Azospirillum lipoferum strain 4B generates in vitro a stable phase variant designated 4VI at frequencies of 10(-4) to 10(-3) per cell per generation. Variant 4VI displays pleitropic modifications, such as the loss of swimming motility and the inability to assimilate certain sugars compared to the wild type. The mechanism underlying phase variation is unknown. To determine whether RecA-mediated processes are involved in phase variation, the recA gene of A. lipoferum 4B was cloned and sequenced and a recA mutant (termed 4BrecA) was constructed by allelic exchange. Strain 4BrecA showed increased sensitivity to UV and MMS compared with 4B and impaired recombinase activity. The ability to generate variants in vitro was not altered; the variants from 4BrecA exhibited all morphological and biochemical features characteristic of the variant generated by strain 4B. However, the frequency of variants generated by 4BrecA was increased by up to 10-fold. So, in contrast with many studies showing the abolition or a large reduction of the frequency of phase variation in recA mutants, this study describes an enhancement of phase variation in the absence of a functional recA.     

Cloning of the Serratia marcescens recA gene and construction of a Serratia marcescens recA mutant

A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.     

Functional properties of Borrelia burgdorferi recA

Functions of the Borrelia burgdorferi RecA protein were investigated in Escherichia coli recA null mutants. Complementation with B. burgdorferi recA increased survival of E. coli recA mutants by 3 orders of magnitude at a UV dose of 2,000 microJ/cm(2). The viability at this UV dose was about 10% that provided by the homologous recA gene. Expression of B. burgdorferi recA resulted in survival of E. coli at levels of mitomycin C that were lethal to noncomplemented hosts. B. burgdorferi RecA was as effective as E. coli RecA in mediating homologous recombination in E. coli. Furthermore, E. coli lambda phage lysogens complemented with B. burgdorferi recA produced phage even in the absence of UV irradiation. The level of phage induction was 55-fold higher than the level in cells complemented with the homologous recA gene, suggesting that B. burgdorferi RecA may possess an enhanced coprotease activity. This study indicates that B. burgdorferi RecA mediates the same functions in E. coli as the homologous E. coli protein mediates. However, the rapid loss of viability and the absence of induction in recA expression after UV irradiation in B. burgdorferi suggest that recA is not involved in the repair of UV-induced damage in B. burgdorferi. The primary role of RecA in B. burgdorferi is likely to be a role in some aspect of recombination.     

Molecular cloning and characterization of the recA gene of Methylomonas clara and construction of recA deficient mutant

Summary The recA gene of the methylotrophic bacterium Methylomonas clara has been isolated from a genomic library by hybridization with the Escherichia coli recA gene. Its complete nucleotide sequence consists of 1029 bp encoding a polypeptide of 342 amino acids. Nucleotide sequence analysis of the M. clara recA gene revealed extensive homologies to recA genes from E. coli and Pseudomonas aeruginosa. Part of the physiological activity of the M. clara RecA protein has become evident in that E. coli recA mutant HB101 is complemented. The cloned recA gene has been modified in vitro by site-specific mutagenesis and by insertion of a kanamycin-resistance gene cassette into the recA coding sequence. M. clara recA mutants were obtained by replacement of the active recA gene by an in-vitro inactivated gene copy. Offprint requests to: K. Esser     

Conditional lethality of the recA441 and recA730 mutants of Escherichia coli deficient in DNA polymerase I

E. coli strains bearing the recA441 mutation and various mutations in the polA gene resulting in enzymatically well-defined deficiencies of DNA polymerase I have been constructed. It was found that the recA441 strains bearing either the polA1 or polA12 mutation causing deficiency of the polymerase activity of pol I are unable to grow at 42 degrees C on minimal medium supplemented with adenine, i.e., when the SOS response is continuously induced in strains bearing the recA441 mutation. Under these conditions the inhibition of DNA synthesis is followed in recA441 polA12 by DNA degradation and loss of cell viability. A similar lethal effect is observed with the recA730 polA12 mutant. The recA441 strain bearing the polA107 mutation resulting in the deficiency of the 5'-3' exonuclease activity of pol I shows normal growth under conditions of continuous SOS response. We postulate that constitutive expression of the SOS response leads to an altered requirement for the polymerase activity of pol I.     

耐辐射奇球菌recA基因的克隆、表达及其对recA缺损大肠杆菌辐射抗性的影响

将耐辐射奇球菌(Deinococcus radiodurans)recA基因克隆到表达质粒pET15b中,并在Escherichia coli HMS中高效表达了可溶性的RecA重组蛋白。同时将recA基因通过穿梭质粒pRADZ3导入recA缺损E.coli TG2细胞中,Western印迹实验显示RecA蛋白能够在不需要诱导剂IPTG的条件下稳定表达。辐射抗性实验表明,D.radiodurans的recA基因在E.coli细胞中的表达能够完全补偿recA缺损E.coli辐射抗性能力。