Action of RuvAB at replication fork structures.

The replicative apparatus often encounters blocks to its progression that necessitate removal of the block and reloading of the replication machinery. In Escherichia coli, a major pathway of replication restart involves unwinding of the stalled fork to generate a four-stranded Holliday junction, which can then be cleaved by the RuvABC helicase-endonuclease. This fork regression may be catalyzed by RecG but is thought to occur even in its absence. Here we test whether RuvAB helicase can also catalyze the unwinding of forked DNA to form Holliday junctions. We find that fork DNA is unwound in the direction required for Holliday junction formation only if the loading of RuvB is restricted to the parental duplex DNA arm. If the binding of RuvB is unrestricted, then RuvAB preferentially unwinds forks in the opposite direction. This is probably related to the greater efficiency of two opposed RuvB hexamers operating across a junction compared with a single hexamer. These data argue against RuvAB acting directly at damaged replication forks and imply that other mechanisms must operate in vivo to catalyze Holliday junction formation.

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.

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.

Seroprevalence of Helicobacter pylori infection in patients with lymphoma.

To determine the Helicobacter pylori (HP) seroprevalence in patients with non-Hodgkin's lymphoma (NHL) and other hematological conditions. Sera were collected from 444 patients with NHL, Hodgkin's disease (HD), lymphoproliferative disorders (LPD), myeloproliferative disorders (MPD), and other hematological conditions. HP seropositivity was determined by ELISA and the results were compared among diagnostic groups HP seropositivity was observed in 168/444 (38%) of the total population. Higher seropositivity rates were associated with increasing age (p=0.001), and country of birth outside the USA and Canada (p=0.0001). Among the diagnostic groups, patients with NHL demonstrated the highest frequency (43%) and those with HD, the lowest frequency (20%; p=.026) of HP seropositivity. The differences among diagnostic groups remained statistically significant after controlling for country of birth (p<0.05), but not after controlling for patient age at diagnosis. The HP seroprevalence of G1 NHL was 55% compared to 40% for non-G1 NHL (p=NS). The highest rate of HP seropositivity (67%) occurred in gastric MALT lymphoma patients, although this did not reach statistical significance compared to the non MALT group (50%) due to small sample size. In conclusion, the rate of HP seropositivity in patients with MALT lymphoma in the USA appears to be lower than in Europe. Helicobacter pylori does not appear to be an important factor in other types of NHL of the G1 tract or elsewhere. Studies of HP prevalence should be controlled for country of birth as well as for age.

High-efficiency transient transfection of endothelial cells for functional analysis.

The definition of signaling pathways in endothelial cells has been hampered by the difficulty of transiently transfecting these cells with high efficiency. This investigation was undertaken to develop an efficient technique for the transfection of endothelial cells for functional analyses. Cells cotransfected with plasmid expressing green fluorescent protein (GFP) and the plasmid of interest were isolated by fluorescence-activated cell sorting (FACS) based on GFP expression. In the sorted cell population, a 2.5-fold enhancement in the number of cells expressing the gene of interest was observed, as confirmed by FACS analysis and Western blotting. Sorted cells retained functional properties, as demonstrated by chemotaxis to the agonist sphingosine 1-phosphate (SPP). To demonstrate the usefulness of this method for defining cellular signaling pathways, cells were cotransfected with plasmids encoding GFP and the carboxyl-terminal domain of the beta-adrenergic receptor kinase (beta ARKct), which inhibits signaling through the beta gamma dimer of heterotrimeric G-proteins. SPP-induced chemotaxis in sorted cells coexpressing beta ARKct was inhibited by 80%, demonstrating that chemotaxis was driven by a beta gamma-dependent pathway. However, no significant inhibition was observed in cells transfected with betaARKct but not enriched by sorting. Thus, we have developed a method for enriching transfected cells that allows the elucidation of crucial mechanisms of endothelial cell activation and function. This method should find wide applicability in studies designed to define pathways responsible for regulation of motility and other functions in these dynamic cells.

Characterisation of the catalytically active form of RecG helicase.

Replication of DNA is fraught with difficulty and chromosomes contain many lesions which may block movement of the replicative machinery. However, several mechanisms to overcome such problems are beginning to emerge from studies with Escherichia coli. An important enzyme in one or more of these mechanisms is the RecG helicase, which may target stalled replication forks to generate a four-stranded (Holliday) junction, thus facilitating repair and/or bypass of the original lesion. To begin to understand how RecG might catalyse regression of fork structures, we have analysed what the catalytically active form of the enzyme may be. We have found that RecG exists as a monomer in solution as measured by gel filtration but when bound to junction DNA the enzyme forms two distinct protein-DNA complexes that contain one and two protein molecules. However, mutant inhibition studies failed to provide any evidence that RecG acts as a multimer in vitro. Additionally, there was no evidence for cooperativity in the junction DNA-stimulated hydrolysis of ATP. These data suggest that RecG functions as a monomer to unwind junction DNA, which supports an 'inchworm' rather than an 'active rolling' mechanism of DNA unwinding. The observed in vivo inhibition of wild-type RecG by mutant forms of the enzyme was attributed to occlusion of the DNA target and correlates with the very low abundance of replication forks within an E.COLI: cell, even during rapid growth.

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.

DNA structure specificity of Rap endonuclease.

The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates.

RecG helicase activity at three- and four-strand DNA structures.

The RecG helicase of Escherichia coli is necessary for efficient recombination and repair of DNA in vivo and has been shown to catalyse the unwinding of DNA junctions in vitro. Despite these findings, the precise role of RecG remains elusive. However, models have been proposed in which RecG promotes the resolution of linked duplexes by targeting three-strand junctions present at D-loops. One such model postulates that RecG catalyses the formation of four-strand (Holliday) junctions from three-strand junctions. To test this model, the DNA binding and unwinding activities of RecG were analysed using synthetic three- and four-strand junctions. The substrate specificity of RecG was found to depend critically on the concentrations of ATP and MgCl(2)and under certain conditions RecG preferentially unwound three-strand junction DNA. This was at least partly due to the larger inhibitory effect of MgCl(2)on the binding of four-strand as opposed to three-strand junctions by RecG. Thus RecG may be targeted to three-strand junctions in vivo whilst still being able to branch migrate the four-strand junctions formed as a result of the initial helicase reaction. The increase in the dissociation constant of RecG on conversion of a three-strand into a four-strand junction may also facilitate resolution of the four-strand junction by the RuvABC complex.

The LH1-RC core complex of Rhodobacter sphaeroides: interaction between components, time-dependent assembly, and topology of the PufX protein.

Mutant strains of the photosynthetic bacterium Rhodobacter sphaeroides, lacking either LH1, the RC or PufX, were analysed by mild detergent fractionation of the cores. This reveals a hierarchy of binding of PufX in the order RC:LH1 > LH1 > RC. The assembly of photosynthetic membranes was studied by switching highly aerated cells to conditions of low aeration in the dark. The RC-H subunit appears before other components, followed by the pufBALMX then pufBA transcripts. Synthesis of the PufX polypeptide precedes that of LH1alpha and beta, which suggests that PufX associates with a limited amount of LH1alpha, beta and the RC, and prior to the encirclement of the RC by the rest of the LH1 complex. The topology of PufX within the intracytoplasmic membrane was determined by proteolytic treatment of membrane vesicles followed by protein sequencing; PufX is N-terminally exposed on the cytoplasmic surface of the photosynthetic membrane.