178,863 research outputs found
Growth phase variation in cell and nucleoid morphology in a Bacillus subtilis recA mutant
No full textThe major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth, Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for addB and recF mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (approximately 15%) of abnormally long cells and nucleoids developed in B. subtilis recA mutants. In addition, recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation
The molecular characterisation of the recA locus in the opportunistic pathogen Bacteroides fragilis
No full textBacteroides fragilis is a human gut commensal and an opportunistic pathogen causing anaerobic abscesses and bacteraemias which are treated with the drug, metronidazole, a DNA damaging agent. The RecA protein is thought to be involved in the repair of metronidazole damage as well as damage caused by oxidative stress. The ability to survive oxygen stress is a strong indicator in an anaerobic bacterium of pathogenic potential and bacterial persistence in the oxygen rich peritoneal cavity. The aim of this thesis was to characterise the B. fragilis recA gene cluster with respect to its genomic context and the transcriptional regulation of the genes in response to metronidazole and oxygen stress. The possible functional roles of the proteins encoded by these genes in protection against these processes would also be evaluated. The functional characterisation of the RecA protein from B. fragilis showed that it was important for survival after exposure to nitrogen (metronidazole) and oxygen (hydrogen peroxide) radicals. RecA was shown to be important for the maintanence of genomic integrity even under normal growth conditions, and overexpression of this protein was shown to be important for increased survival after exposure to metronidazole. RT-PCR of B. fragilis cDNA showed that the recA gene was co-transcribed as an operon together with two upstream genes. Bioinformatic analysis revealed that the first ORF, BF638R1248, was a putative saccharopine dehydrogenase gene (sdh), encoding the SDH protein which may be involved in lysine degradation. The second ORF, BF638R1246/7 had homology to bcp genes, and encoded a putative Bactoferritin co-migratory protein (BCP) belonging to the thiol specific antioxidant superfamily. The functional roles of these proteins suggested that they might also be involved in survival after univalent electron stress. Quantitative RT-PCR showed that all three genes were transcriptionally regulated, but at different levels, after exposure to either metronidazole or H2O2. This suggests that in addition to being expressed as an operon, the genes may also possess independent regulatory elements. Functional characterisation of sdh and bcp was done using a gene mutation approach. Both insertional and deletion mutation methods were attempted but neither produced viable mutants in either of the upstream genes, suggesting that they may be critical for the survival of B. fragilis under normal growth conditions. The use of heterologous gene expression was subsequently employed to establish the functional role of the bcp gene and the encoded putative BCP. A similar approach for SDH was not successful. Heterologous complementation and protein expression of BCP in E. coli, with subsequent biochemical assay, showed that the B. fragilis bcp gene encoded a functional bacterioferritin co-migratory protein (BCP), which is a small thiol-specific protein with antioxidant properties. This BCP showed flexibility in its substrate preference with activity against H2O2, tet-butyl hydroperoxide and linoleic acid. The peroxidase activity of this TSA protein was dependent on the presence of one or more members of the thioredoxin group and NADPH. The BCP aided protection of the enzymatic activity of the B. fragilis redox sensitive Fe-S metalloenzyme Glutamine synthetase (GSIII) during exposure to 100 μM H2O2. There was also evidence to suggest that it aided the recovery of the enzymatic activity of GSIII after exposure to 100 μM H2O2. The findings of this research have resulted in the following hypothesis: The recA operon of B. fragilis acts during host invasion to contribute to the maintenance of DNA integrity and the anaerobic cellular environment until the oxyR regulated system and the pathogenicity genes are activated
Sequence alignment of RecA family proteins from ( RadA), (RadA), (Rad51), (Rad51 and Dmc1), (Dmc1 and ScRad51) and (RecA)
No full text<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins"</p><p></p><p>Nucleic Acids Research 2007;35(6):1787-1801.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874592.</p><p>© 2007 The Author(s)</p> All these RecA-like strand exchange proteins have similar N-terminal domains. The C-terminal RecA domains have been removed for clarity. Secondary structural features of the left-handed RadA helical filament are indicated in cyan (α helices) and red (β strands). Functional motifs are indicated under their corresponding amino acid sequences: the putative dsDNA binding HhH motif, the putative ssDNA binding L1 and L2 loops, the ATP binding Walker A and B motifs, the polymerization motif (PM), the subunit rotation motif (SRM), and others. Positions of the R–E–E triad are indicated using blue arrows
Two time constants for the binding of proteins to DNA from micromechanical data
No full textRecent experimental advances allow the direct measurement of the force/extension behavior for DNA in the presence of strongly binding proteins. Such experiments reveal information about the cooperative mechanism of protein binding. We have studied the irreversible binding of such proteins to DNA using a simple simulation and present a method for estimating quantitative rate constants for the nucleation and growth of linear domains of proteins bound to DNA. Such rate constants also give information about the relative energetics of the two binding processes. We discuss our results in the context of recent data for the DNA-recA-ATPγs system, for which the nucleation time is 4.7 × 104 min per recA binding site and the total growth rate of each domain is 1400 recA/min
Characterizing RecA-Independent Induction of Shiga toxin2-encoding Phages by EDTA Treatment
No full textThe bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA
Functional Characterization of Excision Repair and RecA-Dependent Recombinational DNA Repair in Campylobacter jejuni
No full textThe presence and functionality of DNA repair mechanisms in Campylobacter jejuni are largely unknown. In silico analysis of the complete translated genome of C. jejuni NCTC 11168 suggests the presence of genes involved in methyl-directed mismatch repair (MMR), nucleotide excision repair, base excision repair (BER), and recombinational repair. To assess the functionality of these putative repair mechanisms in C. jejuni, mutS, uvrB, ung, and recA knockout mutants were constructed and analyzed for their ability to repair spontaneous point mutations, UV irradiation-induced DNA damage, and nicked DNA. Inactivation of the different putative DNA repair genes did not alter the spontaneous mutation frequency. Disruption of the UvrB and RecA orthologues, but not the putative MutS or Ung proteins, resulted in a significant reduction in viability after exposure to UV irradiation. Assays performed with uracil-containing plasmid DNA showed that the putative uracil-DNA glycosylase (Ung) protein, important for initiation of the BER pathway, is also functional in C. jejuni. Inactivation of recA also resulted in a loss of natural transformation. Overall, the data indicate that C. jejuni has multiple functional DNA repair systems that may protect against DNA damage and limit the generation of genetic diversity. On the other hand, the apparent absence of a functional MMR pathway may enhance the frequency of on-and-off switching of phase variable genes typical for C. jejuni and may contribute to the genetic heterogeneity of the C. jejuni population
Structure of the hDmc1-ssDNA filament reveals the principles of its architecture
No full textIn eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination
Regulation of expression and nucleotide sequence of the Anabaena variabilis recA gene
No full textThe expression of the cyanobacterial recA gene, isolated from Anabaena variabilis, has been examined at the levels of transcript and protein abundance. Exposure of the cyanobacterium to a variety of DNA-damaging agents, including mitomycin C, methyl methanesulfonate, and UV irradiation, results in a rapid increase in the abundance of the recA transcript above basal levels as determined by Northern (RNA) blot analysis. A concomitant increase in the abundance of a 37- to 38-kilodalton polypeptide was also detected by Western (immuno-) blot analysis of soluble cyanobacterial polypeptides using polyclonal antiserum directed against the Escherichia coli recA protein. The cyanobacterial polypeptide is of the same molecular mass as that synthesized by an in vitro, DNA-directed procaryotic transcription-translation system primed with an A. variabilis genomic fragment containing the recA gene. Nucleotide sequence analysis of the cyanobacterial gene revealed a protein of 358 amino acids with a molecular weight of 38,403 daltons. The A. variabilis and E. coli recA genes share similarity at 58% of the amino acid residues; however, an E. coli-like lexA repressor-binding site is not present in the A. variabilis promoter region. The similarities of A. variabilis and E. coli recA expression and gene sequence are discussed
Molecular cloning of a recA-like gene from the cyanobacterium Anabaena variabilis
No full textA recA-like gene isolated from the cyanobacterium Anabaena variabilis was cloned and partially characterized. When introduced into Escherichia coli recA mutants, the 7.5-kilobase-pair plasmid-borne DNA insert restored resistance to methyl methanesulfonate and UV irradiation, as well as recombination proficiency when measured by Hfr-mediated conjugation. The cyanobacterial recA gene restored spontaneous but not mitomycin C-induced prophage production. Restriction analysis and subcloning yielded a 1.5-kilobase-pair Sau3A fragment which also restored methylmethane sulfonate resistance and coded for a 38- to 40-kilodalton polypeptide when expressed in an in vitro transcription-translation system
CHARACTERIZATION OF RECA GENES AND RECA MUTANTS OF RHIZOBIUM-MELILOTI AND RHIZOBIUM-LEGUMINOSARUM BIOVAR VICIAE
No full textSelbitschka W, Arnold W, PRIEFER UB, et al. CHARACTERIZATION OF RECA GENES AND RECA MUTANTS OF RHIZOBIUM-MELILOTI AND RHIZOBIUM-LEGUMINOSARUM BIOVAR VICIAE. MOLECULAR & GENERAL GENETICS. 1991;229(1):86-95.DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA- Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 bp coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA- mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains
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