Robert G. Lloyd

Modulation of RNA Polymerase by (p)ppGpp Reveals a RecG-Dependent Mechanism for Replication Fork Progression

We have discovered a correlation between the ability of Escherichia coli cells to survive damage to DNA and their ability to modulate RNA polymerase via the stringent response regulators, (p)ppGpp. Elevation of (p)ppGpp, or certain mutations in the beta subunit of RNA polymerase, dramatically improve survival of UV-irradiated strains lacking the RuvABC Holliday junction resolvase. Increased survival depends on excision and recombination proteins and relies on the ability of RecG helicase to form Holliday junctions from replication forks stalled at lesions in the DNA and of PriA to initiate replication restart. The role of RecG provides novel insights into the interplay between transcription, replication, and recombination, and suggests a general model in which recombination underpins genome duplication in the face of frequent obstacles to replication fork progression.

Crystal structure of DNA recombination protein RuvA and a model for its binding to the Holliday junction.

The Escherichia coli DNA binding protein RuvA acts in concert with the helicase RuvB to drive branch migration of Holliday intermediates during recombination and DNA repair. The atomic structure of RuvA was determined at a resolution of 1.9 angstroms. Four monomers of RuvA are related by fourfold symmetry in a manner reminiscent of a four-petaled flower. The four DNA duplex arms of a Holliday junction can be modeled in a square planar configuration and docked into grooves on the concave surface of the protein around a central pin that may facilitate strand separation during the migration reaction. The model presented reveals how a RuvAB-junction complex may also accommodate the resolvase RuvC.

Rescue of stalled replication forks by RecG: Simultaneous translocation on the leading and lagging strand templates supports an active DNA unwinding model of fork reversal and Holliday junction formation

Modification of damaged replication forks is emerging as a crucial factor for efficient chromosomal duplication and the avoidance of genetic instability. The RecG helicase of Escherichia coli, which is involved in recombination and DNA repair, has been postulated to act on stalled replication forks to promote replication restart via the formation of a four-stranded (Holliday) junction. Here we show that RecG can actively unwind the leading and lagging strand arms of model replication fork structures in vitro. Unwinding is achieved in each case by simultaneous interaction with and translocation along both the leading and lagging strand templates at a fork. Disruption of either of these interactions dramatically inhibits unwinding of the opposing duplex arm. Thus, RecG translocates simultaneously along two DNA strands, one with 5′-3′ and the other with 3′-5′ polarity. The unwinding of both nascent strands at a damaged fork, and their subsequent annealing to form a Holliday junction, may explain the ability of RecG to promote replication restart. Moreover, the preferential binding of partial forks lacking a leading strand suggests that RecG may have the ability to target stalled replication intermediates in vivo in which lagging strand synthesis has continued beyond the leading strand.

Dissociation of synthetic Holliday junctions by E.coli RecG protein

The RecG protein of Escherichia coli is needed for normal levels of recombination and for repair of DNA damaged by ultraviolet light, mitomycin C and ionizing radiation. The true extent of its involvement in these processes is masked to a large degree by what appears to be a functional overlap with the products of the three ruv genes. RuvA and RuvB act together to promote branch migration of Holliday junctions, while RuvC catalyses the resolution of these recombination intermediates into viable products by endonuclease cleavage. In this paper, we describe the overproduction and purification of RecG and demonstrate that the overlap extends to the biochemistry. We show that the 76 kDa RecG protein is a DNA‐dependent ATPase, like RuvB. Using gel retardation assays we demonstrate that it binds specifically to a synthetic Holliday junction, like RuvA and RuvC. Finally, we show that in the presence of ATP and Mg2+, RecG dissociates these junctions to duplex products, like RuvAB. We suggest that RecG and RuvAB provide alternative activities than can promote branch migration of Holliday junctions in recombination and DNA repair.

Reverse branch migration of holliday junctions by RecG protein: A new mechanism for resolution of intermediates in recombination and DNA repair

The RecG protein of E. coli is a junction-specific DNA helicase involved in recombination and DNA repair. The function of the protein was investigated using an in vitro recombination reaction catalyzed by RecA. We show that RecG counters RecA-driven strand exchange by catalyzing branch migration of the Holliday junction in the reverse direction. This activity represents a new mechanism for resolving recombination intermediates that is independent of junction cleavage. We discuss how reverse branch migration can facilitate DNA repair, promote recombination in conjugational crosses, and confine the distribution of Chi-stimulated cross-overs. We suggest that the RecG mechanism for resolution of junctions is universal and provides a simple system that allows gene conversion without associated crossing over.

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein–DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression.

Genome stability and the processing of damaged replication forks by RecG

Chromosomal duplication faces many blocks to replication fork progression that could destabilize the genome and prove fatal if not overcome. Overcoming such blocks requires interplay between DNA replication, recombination and repair. The RecG protein of Escherichia coli promotes rescue of damaged forks by catalysing their unwinding and conversion to Holliday junctions. Subsequent processing of this structure allows repair or bypass of the fork block, enabling replication to resume without recourse to potentially mutagenic translesion synthesis or recombination. Such direct rescue of stalled forks might help safeguard genome integrity in all organisms.

Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions.

The formation and subsequent resolution of Holliday junctions are critical stages in recombination. We describe a new Escherichia coli endonuclease that resolves Holliday intermediates by junction cleavage. The 14 kDa Rus protein binds DNA containing a synthetic four‐way junction (X‐DNA) and introduces symmetrical cuts in two strands to give nicked duplex products. Rus also processes Holliday intermediates made by RecA into products that are characteristic of junction resolution. The cleavage activity on X‐DNA is remarkably similar to that of RuvC. Both proteins preferentially cut the same two strands at the same location. Increased expression of Rus suppresses the DNA repair and recombination defects of ruvA, ruvB and ruvC mutants. We conclude that all ruv strains are defective in junction cleavage, and discuss pathways for Holliday junction resolution by RuvAB, RuvC, RecG and Rus.

Direct Rescue of Stalled DNA Replication Forks via the Combined Action of PriA and RecG Helicase Activities

The PriA protein of Escherichia coli plays a key role in the rescue of replication forks stalled on the template DNA. One attractive model of rescue relies on homologous recombination to establish a new fork via PriA-mediated loading of the DnaB replicative helicase at D loop intermediates. We provide genetic and biochemical evidence that PriA helicase activity can also rescue a stalled fork by an alternative mechanism that requires manipulation of the fork before loading of DnaB on the lagging strand template. This direct rescue depends on RecG, which unwinds forks and Holliday junctions and interconverts these structures. The combined action of PriA and RecG helicase activities may thus avoid the potential dangers of rescue pathways involving fork breakage and recombination.

Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product.

SummaryMutation of the recN gene of Escherichia coli in a recBC sbcB genetic background blocks conjugational recombination and confers increased sensitivity to UV light and mitomycin C. The basis for this phenotype was investigated by monitoring the properties associated with recN mutations in otherwise wild-type strains. It was established that recN single mutants are almost fully resistant to UV irradiation, and that there is no detectable defect in repair of UV lesions by excision, error-prone, or recombinational mechanisms. However, recN mutations confer sensitivity to mitomycin C and ionizing radiation both in wild-type and recB sbcB strains. The sensitivity to ionizing radiation is correlated with a deficiency in the capacity to repair DNA double-strand breaks by a UV inducible mechanism. Recombinant λ phages that complement the recombination and repair defects of recN recBC sbcB mutants have been identified, and the recN gene has been cloned from these phages into a low copy-number plasmid.