RecF protein targeting to postreplication (daughter strand) gaps I: DNA binding by RecF and RecFR

Abstract In bacteria, the repair of post-replication gaps by homologous recombination requires the action of the recombination mediator proteins RecF, RecO and RecR. Whereas the role of the RecOR proteins to displace the single strand binding protein (SSB) and facilitate RecA loading is clear, how RecF mediates targeting of the system to appropriate sites remains enigmatic. The most prominent hypothesis relies on specific RecF binding to gap ends. To test this idea, we present a detailed examination of RecF and RecFR binding to more than 40 DNA substrates of varying length and structure. Neither RecF nor the RecFR complex exhibited specific DNA binding that can explain the targeting of RecF(R) to post-replication gaps. RecF(R) bound to dsDNA and ssDNA of sufficient length with similar facility. DNA binding was highly ATP-dependent. Most measured Kd values fell into a range of 60–180 nM. The addition of ssDNA extensions on duplex substrates to mimic gap ends or CPD lesions produces only subtle increases or decreases in RecF(R) affinity. Significant RecFR binding cooperativity was evident with many DNA substrates. The results indicate that RecF or RecFR targeting to post-replication gaps must rely on factors not yet identified, perhaps involving interactions with additional proteins.

(same values as presented in Figure1, open circle, 50% transparency). Binding curves represent experiments done in triplicate in both conditions are plotted on the top panel.
GraphPad PRISM was used to fit the binding curves either using a One binding site model (dotted line) or the Hill-slope model (full line). Characteristics of the binding fit generated either using the One binding site or the Hill-slope models are reported in the bottom panel table.
Supporting figure 2: Steps to generate the 50mer circular ssDNA and RecF binding to 5'-phosphorylated dsDNA A Schematic of the key steps used to generate the circular ssDNA internally FAM labeled, and final electrophoresis gel (12% Native TBE) used to verify the circularization by resistance to ExoI degradation. The "+" and "-" indicate that the samples were submitted or not to ExoI degradation prior to loading. The linar iFAM (lss) was loaded in the first line, followed by the ligated iFAM annealed to 20ds (iFAM 20ds), then the iFAM 20ds treated by PolI which generate the iFAM css, and in the last two lines the css and lss treated by ExoI. B The binding of RecF to 20mer 5'phosphoprylated dsDNA (black circle, dot line) was compared to the binding obtained for the 20mer dsDNA non phosphorylated presented in the Figure 1  with a 3' extension varying in length (9, 6 or 3T) or in composition (12N) was tested in presence of 3 mM ATP (E, red shades) and in the presence of 3mM ATP and 5 µM RecR (F, blue shades) and compared to values obtained in Figure 5 with the 20mer ds harboring a 3' extension (12T) represented with 50% transparency here.

Supporting figure 5: Effects of 5¢ phosphoryl groups
Binding is shown to a number of DNA substrates with comparisons shown for substrates with (dotted lines) and without a 5¢ phosphoryl group (solid line), in presence of 3 mM ATP (red) or 3mM ATP and 5µM RecR (blue). Values obtained in Figure 5 with the 20mer ds harboring a 3' or 5' extension (12T) represented with 50% transparency here.
In panel A, the 5¢ phosphoryl group is placed on the end of the 5¢ ssDNA extension. In panel B, the 5¢ phosphoryl group is at the end of the duplex portion of the DNA substrate. The curves represent the Hill slope fit binding model obtained for each condition. If applicable, the parameters derived from the Hill slope fit are indicated. A The quenching intensity observed for a single strand gap formed between two hairpins lacking a ssDNA extension, or with a 5 or 3' unpaired ssDNA extension. B The quenching intensity observed for a circular gapped DNA with 20 bp of dsDNA, with or without unpaired ssDNA extensions.

Supporting figure 7: Binding to substrates presenting a nick or a 1nt gap
Binding to substrates of 31 mer composed of 31 ds with a nick (solid line) or 30bp with gap in the middle (dashed line) was tested in presence of 3 mM ATP (red) or 3mM ATP and 5µM RecR (blue) and compared to the binding observed for the corresponding 31mer dsDNA labeled DNA previously presented in Figure 2 and represented here with 50% transparency.