Two stages of genome reconstitution in <i>Deinococcus radiodurans</i>.

<p>The first stage, extended synthesis-dependent single-strand annealing (ESDSA) is dominated by nuclease and DNA polymerase functions. The second stage is a more conventional RecA-mediated double-strand break repair process focused on the final splicing of large chromosomal segments.</p

Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination

<div><p>The recombination activity of <i>Escherichia coli</i> (<i>E</i>. <i>coli</i>) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A <i>recA</i> gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 <i>recA</i> gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism <i>in vivo</i>. The growth deficiency is alleviated by expression of the functionally robust RecX protein from <i>Neisseria gonorrhoeae</i>. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism.</p></div

The effect of RecX proteins on ATPase activity of RecA variant proteins.

<p>(A) Effects of EcRecX protein. Reactions contained 3 μM RecA variant, 5 μM M13mp18 ssDNA, 0.5 μM SSB, and 3 mM ATP. The ATP hydrolysis by RecA protein was initiated by the addition of ATP and SSB protein. After 7 min, 100 nM of RecX protein was added and hydrolysis reaction was monitored as shown above. (B) The effect of NgRecX protein on ATPase activity of WT RecA and RecA V79L variant proteins. Reactions were carried out as in panel A, with NgRecX substituted for EcRecX protein where noted.</p

RecA protein amino acid residues affected in RecA variants with increased recombination potential.

<p>Three RecA subunits in a RecA-ssDNA nucleoprotein filament (from coordinates provided by Pavletich and coworkers [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005278#pgen.1005278.ref165" target="_blank">165</a>]) are shown, with a surface contour rendering in which each subunit is transparent but differently colored. The path of the ssDNA within the filament is shown by the black helical line. ADP residues are shown in green. The ATPase active site is at the subunit-subunit interface. Three residues at the ATPase active site (K72 on one face and K248 and K250 on the opposing face) are shown in blue. Prominent residues in which amino acid changes bring about enhanced recombination potential are shown in red/orange.</p

Cell growth competition assays.

<p>Assays were carried out as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005278#sec016" target="_blank">Materials and Methods</a>. (A) Two trial competitions. The top trial shows a competition between two wild type cultures, one of which carries the Ara^– mutation. The lower one shows a competition between wild type cells and cells expressing RecA E38K. As is the case for the RecA variants studied here, RecA E38K also confers a growth disadvantage on cells in which it is expressed. Colony counts revealing the % of cells expressing the mutant RecA proteins with enhanced conjugational function are plotted as a function of the daily growth cycle of the experiment. (B) Competitions between cells expressing each of the three variant proteins and wild type cells. Two competitions are shown for each, with the Ara^– mutation either in the mutant or wild type cells. (C) Competitions between wild type cells (red) and cells with a gene expressing NgRecX protein from the normal <i>recX</i> locus on the <i>E</i>. <i>coli</i> chromosome. The cells expressed either the wild type RecA or RecA V79L from the <i>recA</i> locus as indicated. In two cases, a plasmid also expressing the NgRecX protein at higher levels (pEAW947) was included (NgRecX++). (D) Examples of competition plates from an earlier trial, showing the mixtures of red and white colonies before and after the three days of growth cycles.</p

SOS response of RecA variant proteins.

<p>RecA variant strains containing a plasmid expressing GFP under SOS control (utilizing the <i>recN</i> promoter) were grown in LB. Specific fluorescence, defined as measured fluorescence divided by the OD₆₀₀, is shown. (A) The SOS response in cells expressing RecA variant proteins without treatment with any DNA damaging agent. (B) The SOS response in cells expressing RecA variant proteins after induction by adding 0.005 μg/ml ciprofloxacin at 180 minutes. Due to the error inherent in dividing very small numbers, specific fluorescence is not displayed for times prior to 200 and 230 min in panels A and B, respectively.</p

P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality

<div><p>Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders <i>E</i>. <i>coli</i> more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene <i>ref</i>. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to <i>E</i>. <i>coli</i> in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens <i>Neisseria gonorrhoeae</i> and <i>Staphylococcus aureus</i> serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.</p></div

Measurements of RecA filaments of different size categories.

<p>Measurements of RecA filament lengths for each category were carried out as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005278#sec016" target="_blank">Materials and Methods</a>. Lengths are reported in μm.</p><p>Measurements of RecA filaments of different size categories.</p

Effect of EcRecX on RecA and RecA variant filaments.

<p>Quantitation of electron microscopy results. The fraction of observed molecules in each category described in the text and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005278#pgen.1005278.g004" target="_blank">Fig 4</a> are summarized.</p><p>Effect of EcRecX on RecA and RecA variant filaments.</p