Biomedical subjects

M C Whitby

Publications and source records attributed to M C Whitby.

17 recordsLinked to original sources

PubMed2006

Crossover promotion and prevention.

Homologous recombination is an important mechanism for the repair of double-strand breaks in DNA. One possible outcome of such repair is the reciprocal exchange or crossing over of DNA between chromosomes. Crossovers are beneficial during meiosis because, as well as generating genetic diversity, they promote proper chromosome segregation through the establishment of chiasmata. However, crossing over in vegetative cells can potentially result in loss of heterozygosity and chromosome rearrangements, which can be deleterious. Consequently, cells have evolved mechanisms to limit crossing over during vegetative growth while promoting it during meiosis. Here, we provide a brief review of how some of these mechanisms are thought to work.

Animals↗

PubMed2005

Making crossovers during meiosis.

Homologous recombination (HR) is required to promote both correct chromosome segregation and genetic variation during meiosis. For this to be successful recombination intermediates must be resolved to generate reciprocal exchanges or 'crossovers' between the homologous chromosomes (homologues) during the first meiotic division. Crossover recombination promotes faithful chromosome segregation by establishing connections (chiasmata) between the homologues, which help guide their proper bipolar alignment on the meiotic spindle. Recent studies of meiotic recombination in both the budding and fission yeasts have established that there are at least two pathways for generating crossovers. One pathway involves the resolution of fully ligated four-way DNA junctions [HJs (Holliday junctions)] by an as yet unidentified endonuclease. The second pathway appears to involve the cleavage of the precursors of ligated HJs, namely displacement (D) loops and unligated/nicked HJs, by the Mus81-Eme1/Mms4 endonuclease.

Chromosome Segregation↗

PubMed2001

Fission yeast nascent polypeptide-associated complex binds to four-way DNA junctions.

The four-way DNA junction (X-junction) is both a central intermediate of recombination reactions and, in some cases, a controlling element in transcription and the initiation of DNA replication. Many different proteins have been found to bind to X-junctions in a structure-specific manner. In some cases, this ability only reflects the proteins' general predilection for distorted DNAs but in others the interaction is highly specific and usually signifies that the X-junction is the real target for the protein in vivo. Here we identify the Schizosaccharomyces pombe (Sp) nascent polypeptide associated complex (NAC) as a potent binder of X-junction DNA. NAC is highly conserved in eukaryotes and has reported functions in transcription and the targeting of proteins within the cytosol. NAC is composed of alpha and beta subunits. Each SpNAC subunit has the capacity to bind X-junction DNA, but optimal binding depends on a heterodimer of subunits. Competition assays and binding comparisons using a range of different DNA substrates reveal that SpNAC is highly selective for the X-junction structure. By comparative gel electrophoresis we show that the X-junction is held in its open square conformation when bound by SpNAC. Junction binding is inhibited by concentrations of magnesium ions that are sufficient to "stack" the X-junction, suggesting that SpNAC recognises only the open junction structure. Finally, SpNAC can bind to X-junctions that are already bound by a tetramer of the Escherichia coli RuvA protein, indicating that it interacts with only one face of the junction. The possible biological significance of X-junction binding by SpNAC is discussed.

Amino Acid Sequence↗

PubMed2000

Partial suppression of the fission yeast rqh1(-) phenotype by expression of a bacterial Holliday junction resolvase.

A key stage during homologous recombination is the processing of the Holliday junction, which determines the outcome of the recombination reaction. To dissect the pathways of Holliday junction processing in a eukaryote, we have targeted an Escherichia coli Holliday junction resolvase to the nuclei of fission yeast recombination-deficient mutants and analysed their phenotypes. The resolvase partially complements the UV and hydroxyurea hypersensitivity and associated aberrant mitoses of an rqh1(-) mutant. Rqh1 is a member of the RecQ subfamily of DNA helicases that control recombination particularly during S-phase. Significantly, overexpression of the resolvase in wild-type cells partly mimics the loss of viability, hyper-recombination and 'cut' phenotype of an rqh1(-) mutant. These results indicate that Holliday junctions form in wild-type cells that are normally removed in a non-recombinogenic way, possibly by Rqh1 catalysing their reverse branch migration. We propose that in the absence of Rqh1, replication fork arrest results in the accumulation of Holliday junctions, which can either impede sister chromatid segregation or lead to the formation of recombinants through Holliday junction resolution.

Cell Nucleus↗

PubMed2000

The Holliday junction resolvase SpCCE1 prevents mitochondrial DNA aggregation in Schizosaccharomyces pombe.

SpCCE1 (YDC2) from Schizosaccharomyces pombe is a DNA structure-specific endonuclease that resolves Holliday junctions in vitro. To investigate the in vivo function of SpCCE1 we made an Spcce1:ura4+ insertion mutant strain. This strain is viable and, despite being devoid of the Holliday junction resolvase activity that is readily detected in fractionated extracts from wild-type cells, exhibits normal levels of UV sensitivity and spontaneous or UV-induced mitotic recombination. In accordance with the absence of a nuclear phenotype, we show by fluorescence microscopy that a SpCCE1-GFP fusion localises exclusively to the mitochondria of S. pombe. In Saccharomyces cerevisiae the homologue of SpCCE1, CCE1, is known to function in the mitochondria where its role appears to be to remove recombination junctions and thus facilitate mitochondrial DNA segregation. A similar function can probably be attributed to SpCCE1 in S. pombe, since the majority of mitochondrial DNA from the Spcce1::ura4- strain is in an aggregated form apparently due to extensive interlinking of DNA molecules by recombination junctions. Surprisingly, this marked effect on the conformation of mitochondrial DNA results in little or no effect on proliferation or viability of the Spcce1::ura4+ strain. Possible explanations are discussed.

Cell Compartmentation↗

PubMed1998

Substrate specificity of the SpCCE1 holliday junction resolvase of Schizosaccharomyces pombe.

SpCCE1 from Schizosaccharomyces pombe is an endonuclease that resolves Holliday junctions in vitro. SpCCE1 also binds and cleaves a range of other DNAs (Y-junction; flap; and flayed, nicked, and partial duplexes) with varying efficiency. Cleavage sites are always 3' of thymine nucleotides positioned at or close to the branch point or strand interruption. SpCCE1's favored substrate is the X-junction. Up to two dimers of SpCCE1 can bind concurrently to the same X-junction at its crossover point. From mixing experiments of SpCCE1 and the Escherichia coli RuvA protein, we show that each dimer of SpCCE1 binds to a different face of the X-junction and that both are seemingly competent for strand cleavage. We propose that this provides a mechanism whereby SpCCE1 can scrutinize all four junction strands simultaneously for cleavable thymine nucleotides. SpCCE1 appears to resolve X-junctions by a nick and counter-nick mechanism. Therefore, to ensure a high probability of bilateral strand cleavage, SpCCE1 has a relatively long lifetime on X-junctions. This mechanism has the drawback of limiting dissociation from noncleavable junctions. We discuss why this might not be a problem in vivo.

Base Sequence↗

PubMed1998

Targeting Holliday junctions by the RecG branch migration protein of Escherichia coli.

The RecG protein of Escherichia coli is a junction-specific DNA helicase that drives branch migration of Holliday intermediates in genetic recombination and DNA repair. The reaction was investigated using synthetic X-junctions. RecG dissociates X-junctions to flayed duplex products, although DNA unwinding of the heterologous arms is limited to </=30 base pairs. Junction unwinding requires Mg2+ and the hydrolysis of ATP. X-junction DNA stimulates the ATPase activity of RecG. ATPase activity is also stimulated by linear duplex DNA, although to a lesser extent than by X-DNA, but not by linear single-stranded DNA. In situ 1,10-phenanthroline-copper footprinting shows that RecG binds to the strand cross-over point at the center of the X-junction. Substrate recognition by RecG was investigated using DNAs that represented the various component parts of an X-junction. The minimal DNA structure that RecG forms a stable complex with is a flayed duplex, suggesting that this is the critical feature for junction recognition by RecG. Junction binding and unwinding also depend critically on the concentration of free Mg2+, excess free cation dramatically inhibiting both processes. These inhibitory effects are not mediated specifically by Mg2+; e.g. both Ca2+ and hexamminecobalt(III) chloride also inhibit X-junction binding and unwinding by RecG. The relative abilities of these cations to inhibit RecG-junction binding is correlated with their respective abilities to stack X-junction DNA. From this we conclude that RecG is unable to bind or binds very poorly to fully stacked X-junctions.

Adenosine Triphosphate↗

PubMed1997

A new Holliday junction resolving enzyme from Schizosaccharomyces pombe that is homologous to CCE1 from Saccharomyces cerevisiae.

The resolution of Holliday junctions is a critical stage in recombination. We describe the identification and initial biochemical characterisation of a new Holliday junction resolvase from Schizosaccharomyces pombe. Resolvase activity was initially detected in partially purified cell-free extracts of S. pombe. Resolution of X-junction DNA occurred by the introduction of symmetrical cuts in strands of the same polarity. All cuts occurred 3' of thymine nucleotides with a possible preference for cleavage one nucleotide 3' from the point of strand crossover. During the course of these studies, a potential S. pombe homologue of the Saccharomyces cerevisiae Cruciform Cutting Endonuclease I was identified in the database (SpCCE1). The gene was cloned by PCR, overexpressed in Escherichia coli and its product purified as a His-tagged fusion protein. Purified SpCCE1 binds to X-junctions in a structure-specific manner and resolves them to nicked linear duplex products that are repairable by DNA ligase. SpCCE1 cuts X-junctions in precisely the same way as the resolvase activity from partially purified extracts of S. pombe, indicating that they are probably the same. Finally, we show that SpCCE1 can function as a Holliday junction resolvase in vivo by its ability to complement a resolvase-deficient strain of E. coli.

Bacterial Proteins↗

PubMed1997

Sequence specificity and biochemical characterization of the RusA Holliday junction resolvase of Escherichia coli.

The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates in recombination and DNA repair. Analysis of its subunit structure revealed that the native protein is a dimer. Its resolution activity was investigated using synthetic X-junctions with homologous cores. Resolution occurs by dual strand incision predominantly 5' of CC dinucleotides located symmetrically. A junction lacking homology is not resolved. The efficiency of resolution is related inversely to the number of base pairs in the homologous core, which suggests that branch migration is rate-limiting. Inhibition of resolution at high ratios of protein to DNA suggests that binding of RusA may immobilize the junction point at non-cleavable sites. Resolution is stimulated by alkaline pH and by Mn2+. The protein is unstable in the absence of substrate DNA and loses approximately 80% of its activity within 1 min under standard reaction conditions. DNA binding stabilizes the activity. Junction resolution is inhibited in the presence of RuvA. This observation probably explains why RusA is unable to promote efficient recombination and DNA repair in ruvA+ strains unless it is expressed at a high level.

Base Composition↗

PubMed1996

Interactions between RuvA and RuvC at Holliday junctions: inhibition of junction cleavage and formation of a RuvA-RuvC-DNA complex.

The RuvAB and RuvC enzymes of Escherichia coli define a molecular pathway for the resolution of Holliday intermediates in recombination and DNA repair. They bind specifically to Holliday junctions, and catalyse their branch migration and cleavage, respectively. In a RuvA(B)-junction complex, the Holliday structure is held in an open (square planar) configuration on the concave surface of a 4-fold symmetrical tetramer of RuvA, whereas in a RuvC-junction complex it is folded in an alternative arrangement as part of the cleavage reaction. Genetic studies have shown that the activity of RuvC in vivo depends on RuvAB, which suggests that the two enzymes act in concert, with junction cleavage by RuvC following from branch migration by RuvAB. We have investigated how RuvC can take over a junction from RuvAB to cleave the DNA. We show that RuvA inhibits junction cleavage by RuvC, probably by sandwiching the junction between two tetramers. The extent of inhibition depends on the reaction kinetics of RuvA binding relative to RuvC binding and cleavage. The presence of RuvB and the concentration of Mg2+ both have a significant effect on cleavage in the presence of RuvA. However, a novel protein-DNA complex can be formed when junction DNA is incubated with both RuvA and RuvC. Its mobility is consistent with a RuvC dimer binding to a junction held in an open configuration on the surface of a RuvA tetramer. We suggest that this arrangement provides RuvC with the means to scan the junction during the RuvAB-mediated branch migration reaction for DNA sequences that it can cleave. We further suggest that recognition of the target may provide a trigger for dissociating RuvA, allowing the junction to be folded and cleaved by RuvC.

Bacterial Proteins↗

PubMed1995

Branch migration of three-strand recombination intermediates by RecG, a possible pathway for securing exchanges initiated by 3'-tailed duplex DNA.

RecG protein is required for normal levels of recombination and DNA repair in Escherichia coli. This 76 kDa polypeptide is a junction-specific DNA helicase that acts post-synaptically to drive branch migration of Holliday junction intermediates made by RecA during the strand exchange stage of recombination. To gain further insight into the role of RecG, we studied its activity on three-strand intermediates formed by RecA between circular single-stranded and linear duplex DNAs. Once RecA is removed, RecG drives branch migration of these intermediates by a junction-targeted activity that depends on hydrolysis of ATP. RuvAB has a similar activity. However, when RecG is added to a RecA strand exchange reaction it severely reduces the accumulation of joint molecule intermediates by driving branch migration of junctions in the reverse direction to that catalysed by RecA strand exchange. In comparison, RuvAB has little effect on the reaction. We discuss how reverse branch migration by RecG, which acts counter of the 5'-->3' polarity of RecA binding and strand exchange, could serve to promote or abort the early stages of recombination, depending on the orientation of the single DNA strand initiating the exchange relative to the adjacent duplex region.

Bacterial Proteins↗

PubMed1995

Altered SOS induction associated with mutations in recF, recO and recR.

The SOS system of Escherichia coli aids survival following damage to DNA by promoting DNA repair while cell division is delayed. Induction of the SOS response is dependent on RecA and also on the product of recF. We show that normal induction also requires the products of recO and recR. SOS induction was monitored using a sfiA-lacZ fusion strain. Induction was delayed to a similar degree by mutation in recF, recO or recR. A similar effect was observed following overexpression of RecR from a recombinant recR+ plasmid. We show that the overexpression of RecR also reduces the UV resistance of a recBC sbcBC strain and of a sfiA strain, but not of a rec+sfiA+ strain. The implications of these data for the kinetics of DNA repair are discussed.

Bacterial Proteins↗

PubMed1994

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.

Bacterial Proteins↗

PubMed1994

Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase.

The product of the recG gene of Escherichia coli is needed for normal recombination and DNA repair in E. coli and has been shown to help process Holliday junction intermediates to mature products by catalysing branch migration. The 76 kDa RecG protein contains sequence motifs conserved in the DExH family of helicases, suggesting that it promotes branch migration by unwinding DNA. We show that RecG does not unwind blunt ended duplex DNA or forked duplexes with short unpaired single-strand ends. It also fails to unwind a partial duplex (52 bp) classical helicase substrate containing a short oligonucleotide annealed to circular single-stranded DNA. However, unwinding activity is detected when the duplex region is reduced to 26 bp or less, although this requires high levels of protein. The unwinding proceeds with a clear 3' to 5' polarity with respect to the single strand bound by RecG. Substantially higher levels of unwinding are observed with substrates containing a three-way duplex branch. This is attributed to RecG's particular affinity for junction DNA which we demonstrate would be heightened by single-stranded DNA binding protein in vivo. Reaction requirements for unwinding are the same as for branch migration of Holliday junctions, with a strict dependence on hydrolysis of ATP. These results define RecG as a new class of helicase that has evolved to catalyse the branch migration of Holliday junctions.

Bacterial Proteins↗

PubMed1994

A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions.

The RecG protein of Escherichia coli catalyses branch migration of Holliday junctions made by RecA and dissociates synthetic X junctions into duplex products in reactions that require hydrolysis of ATP. To investigate the mode of action of this enzyme a chromosomal mutation that inactivates recG (recG162) was cloned and sequenced. The recG162 mutation is a G:C to A:T transition, which produces an Ala428 to Val substitution in the protein. This change affects a motif (motif III) in the protein that is highly conserved in DNA and RNA helicases. RecG162 protein was purified and shown to retain the ability to bind synthetic X and Y junctions. However, it does not dissociate these junctions and fails to catalyse branch migration of Holliday junction intermediates purified from a RecA strand exchange reaction. RecG162 retains a DNA-dependent ATPase activity, but this is much reduced relative to the wild-type protein, especially with single-stranded DNA as a co-factor. These results suggest that branch migration by RecG is related to a junction-targeted DNA helicase activity.

Adenosine Triphosphatases↗

PubMed1994

Mutation of recF, recJ, recO, recQ, or recR improves Hfr recombination in resolvase-deficient ruv recG strains of Escherichia coli.

The formation of recombinants in Hfr crosses was studied in Escherichia coli strains carrying combinations of genes known to affect recombination and DNA repair. Mutations in ruv and recG eliminate activities that have been shown to process Holliday junction intermediates by nuclease cleavage and/or branch migration. Strains carrying null mutations in both ruv and recG produce few recombinants in Hfr crosses and are extremely sensitive to UV light. The introduction of additional mutations in recF, recJ, recO, recQ, or recR is shown to increase the yield of recombinants by 6- to 20-fold via a mechanism that depends on recBC. The products of these genes have been linked with the initiation of recombination. We propose that mutation of recF, recJ, recO, recQ, or recR redirects recombination to events initiated by the RecBCD enzyme. The strains constructed were also tested for sensitivity to UV light. Addition of recF, recJ, recN, recO, recQ, or recR mutations had no effect on the survival of ruv recG strains. The implications of these findings are discussed in relation to molecular models for recombination and DNA repair that invoke different roles for the branch migration activities of the RuvAB and RecG proteins.

Crosses, Genetic↗

PubMed1993

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.

Adenosine Triphosphatases↗