Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF-RecO-RecR complex in DNA repair and recombination

Abstract

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF-RecO-RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF-RecO-Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF-RecO-RecR complex functions as an anti-Ssb factor.

Figure 1

Immunoprecipitation of RecF, RecO, and RecR proteins by RecF antibodies. RecF, RecO, and RecR proteins (each at 6 μM) in various combinations were mixed in buffer I containing 35 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, and 100 mM NaCl. RecF antibodies were added, incubated for 30 min on ice followed by the addition of protein A-agarose beads. Samples were then incubated at 4°C with gentle rocking for 10 min. Protein A-agarose-bound antigen–antibody complexes were separated from free proteins by centrifugation at 12,000 × g for 10 min followed by washing three times in buffer I. Samples were resuspended in Laemmli buffer, and proteins were separated on SDS/polyacrylamide gels and visualized by staining with Coomassie blue. Protein combinations used in these experiments are indicated.

Figure 2

Elution profiles of RecF, RecO, and RecR proteins on Superose-12 sizing columns. RecF (1.5 μM) or RecO (6 μM) or RecR (6 μM) in a final volume of 100 μl of buffer I was loaded on Superose-12 HR 10/30 (Pharmacia) and eluted at a flow rate of 0.5 ml/min in the same buffer. Elution time is shown on the x axis and eluted proteins were collected by absorption at 280 nm. For detecting complexes, RecF, RecO, and RecR (each at 6 μM) in the indicated combinations were mixed in buffer I and incubated for 30 min on ice prior to loading on to the column. A, RecF; B, RecO; C, RecR; D, RecO–RecR complex; E, RecF–RecO complex; F, RecF–RecO–RecR complex. The trailing shoulder peak of RecO (B) indicate some interaction of RecO with the resin. The position of molecular mass standards labeled a–e is shown above A and D. a, Ferritin, 400 kDa; b, catalase, 230 k:da; c, aldolase, 130 kDa; d, ovalbumin, 45 kDa; e, chymotrypsin, 25 kDa.

Figure 3

Elution profiles of proteins bound to the Ssb affinity column: RecF (38 μM), RecO (56 μM), and RecR (68 μM) proteins in reaction buffer I were applied to an Ssb protein affinity column preequilibrated with the same buffer I. The column was washed and bound proteins were eluted in a linear gradient of salt to 1 M NaCl. To form complexes proteins as indicated were mixed in the buffer and incubated on ice for 30 min prior to applying on to the column. Total time taken to complete a run is given on the x axis. A, RecF; B, RecO; C, RecR; D, RecF–RecO; E, RecF–RecR. 3F: Peak fractions were analyzed by SDS/PAGE and proteins were visualized by Coomassie blue staining. Fractions corresponding to each peak are indicated. The RecO protein appears to stain less under these conditions.

Figure 4

RecO at 150 μg and RecR at 150 μg, which correspond to 56 μM and 68 μM, respectively, or RecF at 150 μg, RecO at 150 μg, and RecR at 150 μg, which correspond to 38 μM, 56 μM, and 68 μM, respectively, were mixed in buffer I, and incubated on ice for 30 min prior to applying on Ssb affinity column. Bound proteins were eluted in linear gradient of salt. (A) RecO–RecR complex on Ssb affinity column. (B) RecF–RecO–RecR complex on Ssb affinity column. (C) Peak fractions were analyzed by SDS/PAGE and proteins were visualized by Coomassie blue staining. (A) Peak fractions obtained when RecO–RecR was applied to the Ssb column. (B) The peak fraction obtained when the RecF–RecO–RecR complex was applied. Each lane represent proteins present in corresponding peak fractions.

Figure 4

RecO at 150 μg and RecR at 150 μg, which correspond to 56 μM and 68 μM, respectively, or RecF at 150 μg, RecO at 150 μg, and RecR at 150 μg, which correspond to 38 μM, 56 μM, and 68 μM, respectively, were mixed in buffer I, and incubated on ice for 30 min prior to applying on Ssb affinity column. Bound proteins were eluted in linear gradient of salt. (A) RecO–RecR complex on Ssb affinity column. (B) RecF–RecO–RecR complex on Ssb affinity column. (C) Peak fractions were analyzed by SDS/PAGE and proteins were visualized by Coomassie blue staining. (A) Peak fractions obtained when RecO–RecR was applied to the Ssb column. (B) The peak fraction obtained when the RecF–RecO–RecR complex was applied. Each lane represent proteins present in corresponding peak fractions.

Figure 5

Immunoprecipitation of RecF, RecO, RecR, and Ssb protein complexes: RecF, RecO, RecR, and Ssb proteins (each at 6 μM) in indicated combinations in buffer I were incubated on ice for 30 min prior to treating with RecF antibodies. Antigen–antibody mixtures were processed essentially as described in Fig. 1. Order of addition of protein components is indicated. FORSsb indicates that all proteins were incubated together. Ssb before arrow indicates that Ssb protein was added to a preformed RecF–RecO–RecR complex. Similarly R before arrow indicates that RecR protein was added to a complex containing RecF–RecO–Ssb.

Figure 6

RecF interaction with RecO on Ssb affinity column in the presence of ATP: RecF (38 μM) and RecO (56 μM) were mixed in buffer I containing 1 mM ATP and incubated on ice. At the end of the incubation, the sample was applied to Ssb affinity column preequilibrated in buffer I containing 1 mM ATP. Bound proteins were eluted in a linear gradient of salt as described in Fig. 5. The identity of proteins present in the peak fractions was determined by SDS/PAGE and the proteins were visualized by Coomassie blue staining (data not shown).

Figure 7

Cartoon for the RecF–RecO–RecR action in gap repair. For the sake of simplicity only presynaptic events are shown. The first letter of each protein is used to represent the respective protein: S, Ssb; F, RecF; O, RecO; R, RecR; A, 24. (Step 1) gDNA bound by Ssb tetramers (S4). (Step 2) RecF–ATP complex bound at gap junctions of gDNA. It is assumed that RecF protein, in the presence of ATP (step 3) recognizes and subsequently binds at gap junctions. (Step 3) Multiprotein–DNA complexes consisting of RecF–ATP, RecO, RecR, and Ssb. The RecO–RecR are targeted to RecF–ATP–gDNA complexes (step 3) and RecO makes direct contacts with Ssb. (Step 4) Presynaptic filaments consisting of RecF–RecO–RecR–Ssb and RecA proteins. Ssb tetramer displacement is initiated by RecA protein in the presence of RecF–RecO–RecR complex (step 3). Salient features of the cartoon are described in the text.