Recombination and UV resistance of Escherichia coli with the cloned recA and recBCD genes of Serratia marcescens and Proteus mirabilis: evidence for an advantage of intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme

Vries, Johann de; Wackernagel, Wilfried

doi:10.1099/00221287-138-1-31

Cited by 25 publications

(9 citation statements)

References 39 publications

(36 reference statements)

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“…If interactions of SSB with other components of the macromolecular DNA metabolism exist, they seem not to be species specific, because the function of the EcoSSB can be fully replaced by SmaSSB or PmiSSB. In previous experiments, in which other enzymes of the macromolecular DNA metabolism were exchanged between the three species, evidence for species-specific interactions between RecA and RecBCD enzymes was obtained (Rinken et al, 1991;de Vries and Wackernagel, 1992). The tolerance for non-cognate SSB proteins as observed here would also be expected if no interactions of SSB with other enzymes exist.…”

Section: Complementation Of E Coli Mutants By the Ssb Genes Of I! MIsupporting

confidence: 64%

The Single‐stranded‐DNA‐binding Proteins (SSB) of Proteus mirabilis and Serratia marcescens

Vries

Genschel

Urbanke

et al. 1994

European Journal of Biochemistry

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The single-stranded-DNA-binding (SSB) proteins from Proteus mirabilis and Serratia marcescens were purified from overproducing Escherichia coli strains, which were devoid of their own ssb gene. The strains harboured an endA insertion mutation and a xonA mutation resulting in the absence of endonuclease I and exonuclease I activities from the preparations. The amino acid sequences of the SSB of all three species are nearly identical in the N-terminal parts of the proteins that contain the DNA-binding domain, but differ in the C-terminal parts. Both proteins have an apparent binding-site size of 65 and 35 nucleotides at high and low salt concentrations, respectively. The association-rate constant for binding to poly(dT) is 3.2 x 10(8) M-1 s-1 for P. mirabilis SSB (PmiSSB) and 3.4 x 10(8) M-1 s-1 for S. marcescens SSB (SmaSSB). These binding parameters are very similar to those of E. coli SSB (EcoSSB). The structural similarity of the proteins is also documented by the finding that they can exchange subunits among each other to form mixed tetramers. The transcriptional regulation of the ssb and uvrA genes from P. mirabilis and S. marcescens in SOS-induced E. coli cells was studied using lacZ fusions. While the uvrA genes were inducible, there was no induction of the ssb genes transcribed divergently from the uvrA genes. Apparently, regions with nucleotide sequence similarity to the E. coli SOS-box preceding the ssb genes of P. mirabilis and S. marcescens had no gross effect on the transcription. Studies on growth of the cells and recovery from ultraviolet damage indicate that the heterologous SSB proteins support DNA replication and recombinational DNA repair of E. coli with the same efficiency as the E. coli SSB protein. Interactions with other E. coli proteins involved in these processes either do not occur, or are not impeded.

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Section: Complementation Of E Coli Mutants By the Ssb Genes Of I! MIsupporting

confidence: 64%

The Single‐stranded‐DNA‐binding Proteins (SSB) of Proteus mirabilis and Serratia marcescens

Vries

Genschel

Urbanke

et al. 1994

European Journal of Biochemistry

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“…Interestingly, interspecies complementation in vivo demonstrates that optimal recombination is achieved when both RecBCD enzyme and RecA protein are from the same species (Rinken et al 1991;de Vries and Wackernagel 1992). To date, no direct interaction between these enzymes has been detected using either coimmunoprecipitation or biosensor experiments (D.G.…”

Section: Discussionmentioning

confidence: 99%

The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi , resulting in constitutive recombination activation

Churchill¹,

Anderson²,

Kowalczykowski³

1999

Genes & Development

132

123

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Double-strand DNA break repair and homologous recombination in Escherichia coli proceed by the RecBCD pathway, which is regulated by cis-acting elements known as sites. A crucial feature of this regulation is the RecBCD enzyme-directed loading of RecA protein specifically onto the 3-terminal, -containing DNA strand. Here we show that RecBC enzyme (lacking the RecD subunit) loads RecA protein constitutively onto the 3-terminal DNA strand, with no requirement for . This strand is preferentially utilized in homologous pairing reactions. We propose that RecA protein loading is a latent property of the RecBCD holoenzyme, which is normally blocked by the RecD subunit and is revealed following interaction with .

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“…The cells were transformed with a derivative of the single-copy plasmid pRE432 (42), carrying SSB+Gly under the control of the natural SSB promoter (pRE432PROMSSB+Gly, conferring ampicillin resistance). After transformation, the cells were transferred into a 4 ml culture of minimal medium (43) (1× M9 salts, 0.4% glucose, 60 mg l −1 histidine, 230 mg l −1 threonine, 100 mg l −1 proline, 580 mg l −1 arginine, 230 mg l −1 leucine, 170 mg l −1 thymine, 33.7 mg l −1 thiamine, 2 mM MgSO 4 , 0.1 mM CaCl 2 , pH 7.5) supplemented with 40 µg ml −1 ampicillin and 5 µg ml −1 kanamycin and grown overnight at 30°C with heavy shaking.…”

Section: Methodsmentioning

confidence: 99%

Site-directed mutagenesis of the χ subunit of DNA polymerase III and single-stranded DNA-binding protein of E. coli reveals key residues for their interaction

Naue

Fedorov

Pich

et al. 2010

Nucleic Acids Research

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During DNA replication in Escherichia coli, single-stranded DNA-binding protein (SSB) protects single-stranded DNA from nuclease action and hairpin formation. It is known that the highly conserved C-terminus of SSB contacts the χ subunit of DNA polymerase III. However, there only exists a theoretical model in which the 11 C-terminal amino acids of SSB have been docked onto the surface of χ. In order to refine this model of SSB/χ interaction, we exchanged amino acids in χ and SSB by site-directed mutagenesis that are predicted to be of key importance. Detailed characterization of the interaction of these mutants by analytical ultracentrifugation shows that the interaction area is correctly predicted by the model; however, the SSB C-terminus binds in a different orientation to the χ surface. We show that evolutionary conserved residues of χ form a hydrophobic pocket to accommodate the ultimate two amino acids of SSB, P176 and F177. This pocket is surrounded by conserved basic residues, important for the SSB/χ interaction. Mass spectrometric analysis of χ protein cross-linked to a C-terminal peptide of SSB reveals that K132 of χ and D172 of SSB are in close contact. The proposed SSB-binding site resembles those described for RecQ and exonuclease I.

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Recombination and UV resistance of Escherichia coli with the cloned recA and recBCD genes of Serratia marcescens and Proteus mirabilis: evidence for an advantage of intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme

Cited by 25 publications

References 39 publications

The Single‐stranded‐DNA‐binding Proteins (SSB) of Proteus mirabilis and Serratia marcescens

The Single‐stranded‐DNA‐binding Proteins (SSB) of Proteus mirabilis and Serratia marcescens

The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi , resulting in constitutive recombination activation

Site-directed mutagenesis of the χ subunit of DNA polymerase III and single-stranded DNA-binding protein of E. coli reveals key residues for their interaction

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