Loading Of RecA Protein Research Articles

The Escherichia coli DinD Protein Modulates RecA Activity by Inhibiting Postsynaptic RecA Filaments

Escherichia coli dinD is an SOS gene up-regulated in response to DNA damage. We find that the purified DinD protein is a novel inhibitor of RecA-mediated DNA strand exchange activities. Most modulators of RecA protein activity act by controlling the amount of RecA protein bound to single-stranded DNA by affecting either the loading of RecA protein onto DNA or the disassembly of RecA nucleoprotein filaments bound to single-stranded DNA. The DinD protein, however, acts postsynaptically to inhibit RecA during an on-going DNA strand exchange, likely through the disassembly of RecA filaments. DinD protein does not affect RecA single-stranded DNA filaments but efficiently disassembles RecA when bound to two or more DNA strands, effectively halting RecA-mediated branch migration. By utilizing a nonspecific duplex DNA-binding protein, YebG, we show that the DinD effect is not simply due to duplex DNA sequestration. We present a model suggesting that the negative effects of DinD protein are targeted to a specific conformational state of the RecA protein and discuss the potential role of DinD protein in the regulation of recombinational DNA repair.

Regulation of RecA protein by other proteins

The RecA protein is subject to regulation by multiple other proteins in E. coli. These regulatory proteins affect the formation, stability, and function of RecA filaments. The RecFOR proteins are involved in the loading of RecA protein onto SSB‐coated ssDNA. RecOR is necessary and sufficient for this function under most conditions. However, the RecF protein plays varied roles in supplementing the function of RecOR. The RecX protein inhibits RecA protein, and also exhibits a varied palette of inhibitory mechanisms. RecX proteins from different bacteria exhibit the range of potential RecX inhibitory functions. The RecF protein antagonizes RecX function.

SSB Protein Limits RecOR Binding onto Single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.

A RecA Mutant, RecA 730, Suppresses the Recombination Deficiency of the RecBC 1004D–χ* Interaction in Vitro and in Vivo

In Escherichia coli, homologous recombination initiated at double-stranded DNA breaks requires the RecBCD enzyme, a multifunctional heterotrimeric complex that possesses processive helicase and exonuclease activities. Upon encountering the DNA regulatory sequence, χ, the enzymatic properties of RecBCD enzyme are altered. Its helicase activity is reduced, the 3′→5′nuclease activity is attenuated, the 5′→3′ nuclease activity is up-regulated, and it manifests an ability to load RecA protein onto single-stranded DNA. The net result of these changes is the production of a highly recombinogenic structure known as the presynaptic filament. Previously, we found that the recC1004 mutation alters χ-recognition so that this mutant enzyme recognizes an altered χ sequence, χ*, which comprises seven of the original nucleotides in χ, plus four novel nucleotides. Although some consequences of this mutant enzyme–mutant χ interaction could be detected in vivo and in vitro, stimulation of recombination in vivo could not. To resolve this seemingly contradictory observation, we examined the behavior of a RecA mutant, RecA 730, that displays enhanced biochemical activity in vitro and possesses suppressor function in vivo. We show that the recombination deficiency of the RecBC 1004D–χ* interaction can be overcome by the enhanced ability of RecA 730 to assemble on single-stranded DNA in vitro and in vivo. These data are consistent with findings showing that the loading of RecA protein by RecBCD is necessary in vivo, and they show that RecA proteins with enhanced single-stranded DNA-binding capacity can partially bypass the need for RecBCD-mediated loading.

Effects ofrecJ,recQ, andrecFORMutations on Recombination in Nuclease-DeficientrecB recDDouble Mutants ofEscherichia coli

The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5'-->3' exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5'-->3' exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5'-->3' exonuclease is an element of the RecF recombination machinery.