An archaeal RadA paralog influences presynaptic filament formation

Abstract

Recombinases of the RecA family play vital roles in homologous recombination, a high-fidelity mechanism to repair DNA double-stranded breaks. These proteins catalyze strand invasion and exchange after forming dynamic nucleoprotein filaments on ssDNA. Increasing evidence suggests that stabilization of these dynamic filaments is a highly conserved function across diverse species. Here, we analyze the presynaptic filament formation and DNA binding characteristics of the Sulfolobus solfataricus recombinase SsoRadA in conjunction with the SsoRadA paralog SsoRal1. In addition to constraining SsoRadA ssDNA-dependent ATPase activity, the paralog also enhances SsoRadA ssDNA binding, effectively influencing activities necessary for presynaptic filament formation. These activities result in enhanced SsoRadA-mediated strand invasion in the presence of SsoRal1 and suggest a filament stabilization function for the SsoRal1 protein.

Fig. 1. SsoRal1 alters SsoRadA ssDNA-dependent ATP hydrolysis

Assays were performed using the coupled ATPase assay as previously described [20] at 80 °C using 3 μM ssϕX174 as the substrate. The first protein was added prior to time 0 and, where applicable, the second protein was added after 6 min (as indicated by dashed vertical lines). Symbols are: SsoRal1, purple triangles; SsoRadA, green squares; SsoRadA second, blue inverted triangles; SsoRal1 second, black circles; SsoRadA and SsoRal1 added together at time 0, red diamonds. (A) SsoRal1 and SsoRadA used at saturating concentrations (1 μM). (B) SsoRadA at a saturating concentration (1 μM) and SsoRal1 at a subsaturating concentration (0.03 μM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. SsoRal1 and wild-type SsoRadA ssDNA binding characteristics

Representative mobility shift assays are shown. (A) Salt midpoint titration of 1.7 μM SsoRadA, where ssDNA–protein complexes were formed in the presence of salt and then either cross-linked with 0.75% (final concentration) glutaraldehyde (left side of panel) or were not subjected to cross-linking (right side of panel). (B) Salt titration of 1.7 μM SsoRal1, where ssDNA–protein complexes were formed in the presence of salt but did not require cross-linking for visualization. (C) Reactions containing 100 mM NaCl and increasing concentrations of SsoRal1 were incubated with ssDNA in the presence or absence of ATP and without cross-linking. All reactions contained 5 μM nucleotides ssDNA. In all panels the position of the wells and unbound oligonucleotide are indicated.

Fig. 3. SsoRal1 enhances SsoRadA–ssDNA binding

(A) A representative mobility shift assay is shown for SsoRal1 in combination with wild-type SsoRadA. All reactions contained 5 μM ssDNA as the substrate, 100 mM NaCl, and 3 mM ATP. Where used together, SsoRadA and SsoRal1 proteins were added simultaneously. The position of shifted species and unbound oligonucleotide are indicated. (B) Western blot detection of SsoRadA and SsoRal1 in stabilized nucleoprotein filaments. Using a gel identical to that shown in (A), shifted products located in the SsoRadA position (indicated by arrow) in lanes 5 and 6 (the most slowly migrating bands) were directly excised from the gel and subjected to electrophoresis followed by Western blotting. Purified SsoRadA and SsoRal1 proteins were used as controls and protein concentrations are indicated above the lanes.

Fig. 4. Binding of SsoRal1 and SsoRadA to ssDNA is influenced by nucleotide cofactors

EMSAs are shown for SsoRal1 in (A) and wild-type SsoRadA (B) where binding reactions were performed using ATP, ADP, or ATPγS. All reactions included 5 μM ssDNA and 100 mM NaCl. ATP and ADP concentrations were 3 mM, while ATPγS was present at a concentration of 50 μM. All wild-type SsoRadA reactions were cross-linked by the addition of glutaraldehyde to a final concentration of 0.75%. The position of the wells and unbound oligonucleotide are indicated.

Fig. 5. ssDNA binding characteristics of SsoRadA ATPase motif mutants

Representative mobility shift assays are shown for the SsoRadA K120A and K120R mutant proteins. (A) SsoRadA K120A and K120R were incubated in increasing concentrations of NaCl with 5 μM ssDNA in the presence of 3 mM ATP. (B) Cofactors were included in the binding reactions with 100 mM NaCl. ATP and ADP concentrations were 3 mM, while ATPγS was present at a concentration of 50 μM. All reactions included 5 μM ssDNA as the substrate, and were cross-linked by addition of glutaraldehyde to a final concentration of 0.75%. The position of the wells and unbound oligonucleotide are indicated.

Fig. 6. ssDNA binding of SsoRal1 with SsoRadA ATPase motif mutants

A representative mobility shift assay is shown for SsoRal1 in combination with SsoRadA K120A and K120R mutant proteins. All reactions contained 5 μM ssDNA as the substrate, 100 mM NaCl, and 3 mM ATP. The position of shifted species and unbound oligonucleotide are indicated.

Fig. 7. SsoRal1 catalyzes strand invasion and enhances SsoRadA D-loop production

(A) Schematic of the D-loop reaction. (B) Representative autoradiographs showing D-loop formation by various proteins or combinations of proteins at 1, 5, 10, and 20 min. NP is no protein and was taken at 20 min. Concentrations of SsoRadA, SsoRadA K120R, and SsoRadA K120A were 0.6 μM, while concentration of SsoRal1 was 0.009 μM. (C) Quantitation of D-loop formation by individual proteins or protein combinations as indicated by the labels along the X-axis. Error bars represent the standard deviation of a minimum of three repetitions. The amount of D-loop product formed by SsoRadA at 1 min was arbitrarily set at 1, and values for all other bars are compared to the value for SsoRadA at 1 min. The dashed line is set at 1 for ease of comparison across the graph.

Fig. 8. Model of SsoRal1 stabilization of the SsoRadA nucleoprotein filament

(A) The SsoRadA presynaptic filament is dynamic and slow to form. SsoRadA binds ssDNA and dissociates rapidly. (B) and (C) Binding of SsoRal1 to ssDNA reduces dissociation of SsoRadA and stabilizes the filament. (D) Strand invasion is enhanced through reduction of SsoRadA presynaptic filament dynamics due to SsoRal1 binding.