Effects of ATP and primer-template binding on RecA-UmuC cross-linking efficiency in pol V Mut.

<p>Western blot using an antibody to UmuC demonstrating increased UmuC-RecA cross-linking with increasing ATPγS concentration and decreasing UmuC-RecA cross-linking with increasing primer-template concentration in the presence of 500 μM ATPγS. pol V Mut was generated using RecA N113Bpa as described in Methods, with ATPγS and primer-template hairpin DNA added where indicated. ATPγS concentration ranges from 0.8 to 500 μM and primer-template DNA concentration ranges from 0.01–5 μM.</p

RecA D112R and D112R N113R exhibit wild-type RecA binding affinities for pol V.

<p>(A) Location of investigated residues on the RecA protein surface. The D112 and N113 residues compose an acidic knob on the RecA surface. The RecA monomer represented in electrostatic coloring scheme (red = negative charged residues, blue = positive charged residues) was generated in Pymol (PDB 3CMU [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005066#pgen.1005066.ref067" target="_blank">67</a>]). In this illustration, the monomer shown is located at the 3' end of the ssDNA (the 3'-proximal RecA monomer), which is the monomer removed by pol V during the activation cycle. (B) Altering this acidic surface to basic residues does not affect the binding affinity for pol V. Equilibrium binding isotherms of wild-type RecA, RecA D112R, and RecA D112R titrated with pol V as monitored by fluorescence depolarization. All data are the average of at least three experiments. Error bars are one standard deviation from the mean.</p

RecA D112R and RecA D112R N113R promote autocatalytic cleavage of LexA and UmuD <i>in vitro</i>.

<p>Reactions were carried out as described in Methods. At least four repeats with all RecA variants were conducted. Representative gels are shown. (A) Cleavage of UmuD protein. Reactions containing 5 μM of M13mp18 cssDNA, 3 μM RecA, 3 mM ATPγS, and 8 μM UmuD were incubated at 37°C for 0, 15, and 60 minutes and visualized by staining with Coomassie blue. (B) Cleavage of LexA protein. Reactions containing 5 μM of M13mp18 cssDNA, 3 μM RecA, 3 mM ATPγS, and 8 μM LexA were incubated at 37°C for 0, 15, and 30 minutes. The products of the reaction were separated on a 15% SDS-PAGE and visualized as in (A). (C) Quantification of the LexA and UmuD <i>in vitro</i> cleavage.</p

The RecA 3'-surface interacts with UmuC in pol V Mut.

<p>The photo-reactive unnatural amino acid Bpa was incorporated into RecA at position N113 (RecA N113Bpa) and used to probe RecA/pol V Mut interactions. The protocol for the generation of pol V Mut was followed as described in Methods, with ATP, ATPγS, and template-primer added where indicated. Samples were exposed to UV light to covalently crosslink the RecA N113Bpa to nearby interacting partners (A) Coomassie stained SDS-PAGE of samples. UmuC, RecA, and UmuD' bands are indicated. Higher molecular weight bands appear upon cross-linking via UV light. The major cross-linked species runs at ~100 kDa. (B) Western blot of reactions presented in (A) using antibodies to the RecA protein. (C) UmuC western blot of reactions presented in (A). UmuC is present in the major cross-linked band at ~100 kDa. (D) UmuD' western blot of reactions presented in (A). A non-specific band is present at ~49 kDa which is not UV crosslinking dependent. A weak band observed at ~60 kDa was UV-dependent, but was not observed via SDS-PAGE. Therefore, it could not be pursued via mass spectrometry analysis.</p

Analysis of cross-linked RecA-UmuC product #1.

<p>The first of the two identified cross-linked products appeared in samples generated via both active and inactive pol V protocols, although it seemed to be somewhat more prominent in the inactive samples. The UmuC peptide involved in the cross-linking encompasses residues 361–376. MS/MS spectra for each of the three peaks seen in the extracted ion chromatogram (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005066#pgen.1005066.g012" target="_blank">Fig. 12</a>) for the RecA-UmuC cross-linked product are shown at the top. The predicted crosslinking locations for peaks A,B, and C are shown at the bottom. The lettering corresponds to the unique elution profile for the same precursor ion.</p

RumA′₂B can be activated by all RecA variants.

<p>Transactivation reaction was carried out on 3-nt overhang hairpin for UmuD′₂C (A) and RumA′₂B (B). RecA* (400 nM) was preformed on 30 nt ssDNA then mixed with UmuD′₂C or RumA′₂B (400 nM each) and incubated for 30 mins at 37°C. Unlike UmuD′₂C (A), RumA′₂B can be activated by RecA variants D112R, D112R N113R, and S117F (B).</p

RecA variant D112R N113R is defective in DNA polymerase V activation.

<p>(A) Schematic of DNA synthesis by pol V transactivated by RecA*. (B) In pol V reaction buffer, 5 mM DTT, 2 mM ATPγS, 2 μM poly (dT)₅₀, and 20 μM RecA were incubated at 37°C for 10 minutes to allow for formation of RecA*. This was followed by the addition of 1 mM dNTPs, 1 μM pol V, and 110 nM p/t hairpin DNA and incubation for 1 hour at 37°C. During this time pol V Mut is formed, synthesizes DNA, and can be reactivated for another round of synthesis (right panel, +Pol V). Reactions were repeated without the addition of pol V to demonstrate the absence of contaminating polymerase in the RecA preps (left panel,—Pol V).</p

Extracted Ion Chromatograms for the RecA-UmuC cross-linked peptides.

<p>(A) Cross-linked product #1. This was the most prominent cross-linked product and was found in virtually all samples generated with either of the two protocols (active or inactive). The product appeared to be somewhat more prominent in the inactive samples. (B) Cross-linked product #2. This product was more prominent in the active samples. Signal intensity was normalized to the highest for each unique peptide pair within a panel (A or B). However, the panel scales are different. Peaks in panel B are approximately 10 fold less prominent than those in panel A.</p

Structural model for UmuC.

<p>The model was generated by the program Phyre2 (<b>P</b>rotein <b>H</b>omology/analog<b>Y R</b>ecognition <b>E</b>ngine V 2.0) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005066#pgen.1005066.ref079" target="_blank">79</a>] using intensive modeling mode, and exhibits a close structural homology to the known structure of polymerase Dpo4 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005066#pgen.1005066.s002" target="_blank">S2 Fig</a>). The locations of cross-linked peptides 1 (blue) and 2 (green) are highlighted, with particular amino acid residues involved in cross-linking highlighted in red.</p

Defining the step in the pol V activation pathway that is affected by RecA D112R N113R.

<p>Active DNA polymerase V-Mut was generated with multiple RecA variants as described in Methods, and subjected to several experimental procedures. (A) The activity of pol V Mut D112R, pol V Mut N113RBpa and pol V Mut D112R N113R (400 nM each) was detected on 5'-³²P-labeled 12 nt oh HP (100 nM) in the presence or absence of ATP/ATPγS and dNTPs. ssDNA and ATPγS were added to the reactions to detect free RecA and determine whether pol V Mut could be transactivated (left panels). Pol V Mut variants were resolved by SDS-PAGE and protein components were visualized via Imperial staining (right panels). (B) Binding of pol V Mut and pol + RecA mixtures (400 nM each) to ε-ATP (500 μM) was measured using rotational anisotropy. The error bars correspond to 1 SEM. Numbers above four of the bars are described in the text. (C) Binding of pol V Mut WT (400 nM), pol V + RecA D112R N113R (400 nM each) and pol V Mut D112R N113R (400 nM) to fluorescein-labeled 12 nt oh HP (50 nM) was measured as a function of ATPγS. DNA binding was observed as a change in rotational anisotropy. The rotational anisotropy of HP alone is included in the graph.</p