The recombination mediator proteins RecFOR maintain RecA* levels for maximal DNA polymerase V Mut activity

Abstract

DNA template damage can potentially block DNA replication. Cells have therefore developed different strategies to repair template lesions. Activation of the bacterial lesion bypass DNA polymerase V (Pol V) requires both the cleavage of the UmuD subunit to UmuD' and the acquisition of a monomer of activated RecA recombinase, forming Pol V Mut. Both of these events are mediated by the generation of RecA* via the formation of a RecA-ssDNA filament during the SOS response. Formation of RecA* is itself modulated by competition with the ssDNA-binding protein (SSB) for binding to ssDNA. Previous observations have demonstrated that RecA filament formation on SSB-coated DNA can be favored in the presence of the recombination mediator proteins RecF, RecO, and RecR. We show here using purified proteins that in the presence of SSB and RecA, a stable RecA-ssDNA filament is not formed, although sufficient RecA* is generated to support some activation of Pol V. The presence of RecFOR increased RecA* generation and allowed Pol V to synthesize longer DNA products and to elongate from an unpaired primer terminus opposite template damage, also without the generation of a stable RecA-ssDNA filament.

Keywords: DNA damage; DNA polymerase; DNA recombination; DNA repair; RecA; lesion bypass; nucleic acid enzymology; recombination mediator protein; translesion synthesis.

Figure 1.

Requirements for Pol V Mut–catalyzed DNA synthesis and lesion bypass. A, diagram of the primer template. The red asterisk indicates a 5′-[³²P] end label. B, requirements for DNA synthesis. Primer extension assays were as described under “Experimental procedures,” and the DNA products were analyzed by electrophoresis through a 20% polyacrylamide gel containing 7 m urea. Omission indicates the protein omitted from the reaction. Lane (C), the complete reaction; lane V, Pol V; lane β, β clamp; lane S, SSB; lane A, RecA; lane F, RecF; lane O, RecO; lane R, RecR; lane T, RecFOR; EXT (%), the fraction of the primer extended past the template damage; TLS (%), the fraction of the primer extended up to and across from the template damage; UNDAMAGED, undamaged p/t; CPD, p/t carrying a cyclopyrimidine dimer in the template strand in the position shown in A; THF, p/t carrying a THF abasic site analog in the template strand in the position shown in A; p, unextended primer; 102, represents full extension of the primer. C, RecFOR stimulates the synthesis of long DNA products. Shown are densitometric traces of lane 1 (red) and lane 9 (blue) of the experiment with the undamaged p/t. PSL, photostimulated luminescence. D, SDS-PAGE of the protein preparations used in this study. The indicated proteins (2 μg, except the DnaX cx (5 μg) were electrophoresed through a 12% Novex Bis-Tris gel run in MES buffer. The gel was stained with Coomassie Brilliant Blue and photographed.

Figure 2.

SSB is required to observe stimulation of Pol V Mut by RecFOR. A, sample gels comparing the activity of different combinations of RecF, RecO, and RecR as indicated in either the presence or absence of SSB. B, TLS and extension (EXT) as described in the legend to Fig. 1 on the undamaged (UND), CPD, and THF p/ts in the presence and absence of SSB. Median and standard deviation are given for three independent experiments.

Figure 3.

The effect of SSB and RecA on primer extension by Pol IV. Pol IV–catalyzed primer extension with the indicated proteins present was as described under “Experimental procedures” using the undamaged p/t. Analysis was as described above in the legend to Fig. 1.

Figure 4.

Primer accessibility assay. The indicated combinations of proteins were used in primer accessibility assays as described under “Experimental procedures.” Primer degraded (%), the fraction of primer degraded by Exo III in each assay calculated as 1 minus the fraction of total radioactivity in the intact primer band after incubation converted to a percentage.

Figure 5.

RecA730 provides equivalent Pol V Mut activity as the combination of WT RecA and RecFOR. A, primer extension assays with the THF p/t either in the presence of WT RecA or RecA730 and either in the absence of RecFOR or in the presence of RecFOR or RecOR as indicated. Assays were performed and analyzed as described under “Experimental procedures” and in the legend to Fig. 1. B, densitometric traces of lane 1 (blue), lane 5 (black), and lane 6 (red) of the gel shown in A.

Figure 6.

LexA cleavage under Pol V primer extension assay conditions. LexA cleavage reactions were as described under “Experimental procedures.” The assay conditions were identical to those for Pol V primer extension. In addition to the proteins indicated in the figure, reaction mixtures also contained β, DnaX cx, 2 μm NHP-LexA, and [³²P]NHP-LexA. FL, full-length NHP-LexA; CP, N-terminal cleavage product of NHP-LexA.