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

Paromita Raychaudhury,Kenneth J Marians

doi:10.1074/jbc.ra118.005726

Paromita Raychaudhury, Kenneth J Marians

Open Access

https://doi.org/10.1074/jbc.ra118.005726

Copy DOI

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.

Highlights

DNA template damage can potentially block DNA replication
Unlike with RecA, RecFOR had little effect on RecA730-supported polymerase V (Pol V) activity in the presence of ssDNA-binding protein (SSB) (Fig. 5), we suggest that the difference in LexA cleavage mediated by RecA730 compared with RecA in the presence of SSB reports on the extent of RecA* generation required for maintenance of maximal Pol V activity
As judged by the observations that RecA in the absence of SSB strongly inhibits Pol V DNA synthesis activity (Fig. 1) and that this inhibition is obviated when SSB is present, we argue that even in the presence of RecFOR, a stable, exclusively RecA–ssDNA filament does not form

Summary

To whom correspondence should be addressed

Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Ave., New York, NY 10065. The recombination mediator proteins RecF, RecO, and RecR are known to facilitate RecA nucleation on SSB–ssDNA [23] and in gaps [24] It is not surprising, that recFOR mutants display a delay in the induction of the SOS response [25, 26]. That recFOR mutants display a delay in the induction of the SOS response [25, 26] These observations led Fujii et al [27] to examine the roll of the RecFOR proteins in TLS by Pol V. They found, using a primer extension assay, that TLS by Pol V in the presence of SSB, RecA, and the ␤ sliding clamp required the RecFOR proteins They suggested that the RecFOR requirement was a result of mediating RecA filament formation on the ssDNA. Per se but reflects a demand to maintain RecA* levels sufficient for maximal Pol V Mut activity during lesion bypass

Results and discussion

Conclusions

Experimental procedures