Biomedical subjects

Robert G Lloyd

Publications and source records attributed to Robert G Lloyd.

At least 19 recordsLinked to original sources

PubMed2006

RusA Holliday junction resolvase: DNA complex structure--insights into selectivity and specificity.

We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli RusA resolvase bound to a duplex DNA substrate that reveals critical protein-DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The RusA enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, RusA shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA. However, RusA does not show DNA cleavage activity with duplex substrates. Our structure suggests the possible basis for structural selectivity as well as sources of the sequence specificity observed for DNA cleavage by RusA.

Amino Acid Substitution↗

PubMed2006

Rep and PriA helicase activities prevent RecA from provoking unnecessary recombination during replication fork repair.

The rescue of replication forks stalled on the template DNA was investigated using an assay for synthetic lethality that provides a visual readout of cell viability and permits investigation of why certain mutations are lethal when combined. The results presented show that RecA and other recombination proteins are often engaged during replication because RecA is present and provokes recombination rather than because recombination is necessary. This occurs particularly frequently in cells lacking the helicase activities of Rep and PriA. We propose that these two proteins normally limit the loading of RecA on ssDNA regions exposed on the leading strand template of damaged forks, and do so by unwinding the nascent lagging strand, thus facilitating reannealing of the parental strands. Gap closure followed by loading of the DnaB replicative helicase enables synthesis of the leading strand to continue. Without either activity, RecA loads more frequently on the DNA and drives fork reversal, which creates a chickenfoot structure and a requirement for other recombination proteins to re-establish a viable fork. The assay also reveals that stalled transcription complexes are common impediments to fork progression, and that damaged forks often reverse independently of RecA.

Adenosine Triphosphatases↗

PubMed2005

RNA polymerase modulators and DNA repair activities resolve conflicts between DNA replication and transcription.

Organisms rely on close interplay between DNA replication, recombination, and repair to secure transmission of the genome. In rapidly dividing cells, there is also great pressure for transcription, which may induce conflict with replication. We investigated the potential for conflict in bacterial cells, where there is no temporal separation of these processes. Eliminating the stringent response regulators ppGpp and DksA or the GreA and Mfd proteins, which revive or dislodge stalled transcription complexes, and especially combinations of these factors, is shown to severely reduce viability when DNA repair is also compromised. Both ppGpp and certain RNA polymerase (RNAP) mutations reduce accumulation of backed-up arrays of stalled transcription complexes. We propose these arrays are formidable obstacles to replication that are normally kept in check in wild-type cells by ppGpp, DksA, GreA, and Mfd. When arrays do obstruct replication, the consequences are resolved by one of the many pathways available to rescue stalled forks.

DNA Repair↗

PubMed2005

AFM studies on the role of the protein RdgC in bacterial DNA recombination.

Genetic studies of rdgC in different bacterial systems suggest that it may play a role in replication and recombination. However, the exact function of the corresponding protein, RdgC, is unknown. In this study, we have imaged complexes of RdgC with both linear and supercoiled circular plasmid DNA using atomic force microscopy. We confirm that RdgC does not target any specific sequences in double-stranded DNA, as has been suggested from biochemical data. However, we detect an increased affinity of the protein to DNA ends, and an ability to promote bending of DNA. Similar binding preferences have been reported for enzymes involved in recombination. Protein complexes with supercoiled plasmid DNA further enabled us to study the effect of RdgC on DNA superstructure. At high concentrations of protein we observed promotion of DNA condensation. Recombination is largely enhanced by close contacts of distant regions along the DNA strands, as can occur, for instance, through condensation. Our data thus support a possible function of RdgC as a midwife of recombination.

DNA, Bacterial↗

PubMed2005

DNA binding by the substrate specificity (wedge) domain of RecG helicase suggests a role in processivity.

RecG differs from most helicases acting on branched DNA in that it is thought to catalyze unwinding via translocation of a monomer on dsDNA, with a wedge domain facilitating strand separation. Conserved phenylalanines in the wedge are shown to be critical for DNA binding. When detached from the helicase domains, the wedge bound a Holliday junction with high affinity but failed to bind a replication fork structure. Further stabilizing contacts are identified in full-length RecG, which may explain fork binding. Detached from the wedge, the helicase region unwound junctions but had extremely low substrate affinity, arguing against the "classical inchworm" mode of translocation. We propose that the processivity of RecG on branched DNA substrates is dependent on the ability of the wedge to establish strong binding at the branch point. This keeps the helicase motor in contact with the substrate, enabling it to drive dsDNA translocation with high efficiency.

Amino Acid Sequence↗

PubMed2005

Conservation of RecG activity from pathogens to hyperthermophiles.

Maintaining the integrity of the genome is essential for the survival of all organisms. RecG helicase plays an important part in this process in Escherichia coli, promoting recombination and DNA repair, and providing ways to rescue stalled replication forks by way of a Holliday junction intermediate. We purified RecG proteins from three other species: two Gram-positive mesophiles, Bacillus subtilis and Streptococcus pneumoniae, and one extreme thermophile, Aquifex aeolicus. All three proteins bind and unwind replication fork and Holliday junction DNA molecules with efficiencies similar to the E. coli protein. Proteins from the Gram-positive species promote DNA repair in E. coli, indicating either that RecG acts alone or that any necessary protein-protein interactions are conserved. The S. pneumoniae RecG reduces plasmid copy number when expressed in E. coli, indicating that like the E. coli protein it unwinds plasmid R loop structures used to prime replication. This effect is not seen with B. subtilis RecG; the protein either lacks R loop unwinding activity or is compromised by having insufficient ATP. The A. aeolicus protein unwinds DNA well at 60 degrees C but is less efficient at 37 degrees C, explaining its inability to function in E. coli at this temperature. The N-terminal extension present in this protein was investigated and found to be dispensable for activity and thermo-stability. The results presented suggest that the role of RecG in DNA replication and repair is likely to be conserved throughout all bacteria, which underlines the importance of this protein in genome duplication and cell survival.

Bacteria↗

PubMed2005

RecN protein and transcription factor DksA combine to promote faithful recombinational repair of DNA double-strand breaks.

In rapidly dividing bacterial cells, the machinery for repair of DNA double-strand breaks has to contend not only with the forces driving replication and transmission of the DNA but also its transcription. By exploiting I-SceI homing endonuclease to break the Escherichia coli chromosome at one or more defined locations, we have been able to investigate how these processes are co-ordinated and repair is accomplished. When breaks are induced at a single site, the SOS-inducible RecN protein and the transcription factor DksA combine to promote efficient repair. When induced at two or more, distantly located sites, RecN becomes almost indispensable. Many cells that do survive have extensive deletions of sequences flanking the break, with end points often coinciding with imperfect repeat elements. These findings herald a much greater complexity for chromosome repair than suggested by current mechanistic models and reveal a role for RecN in protecting the chromosome from break-induced chromosome rearrangements.

Bacterial Proteins↗

PubMed2004

Interplay between DNA replication, recombination and repair based on the structure of RecG helicase.

Recent studies in Escherichia coli indicate that the interconversion of DNA replication fork and Holliday junction structures underpins chromosome duplication and helps secure faithful transmission of the genome from one generation to the next. It facilitates interplay between DNA replication, recombination and repair, and provides means to rescue replication forks stalled by lesions in or on the template DNA. Insight into how this interconversion may be catalysed has emerged from genetic, biochemical and structural studies of RecG protein, a member of superfamily 2 of DNA and RNA helicases. We describe how a single molecule of RecG might target a branched DNA structure and translocate a single duplex arm to drive branch migration of a Holliday junction, interconvert replication fork and Holliday junction structures and displace the invading strand from a D loop formed during recombination at a DNA end. We present genetic evidence suggesting how the latter activity may provide an efficient pathway for the repair of DNA double-strand breaks that avoids crossing over, thus facilitating chromosome segregation at cell division.

Chromosome Segregation↗

PubMed2004

RecG helicase promotes DNA double-strand break repair.

Double-strand breaks pose a major threat to the genome and must be repaired accurately if structural and functional integrity are to be preserved. This is usually achieved via homologous recombination, which enables the ends of a broken DNA molecule to engage an intact duplex and prime synthesis of the DNA needed for repair. In Escherichia coli, repair relies on the RecBCD and RecA proteins, the combined ability of which to initiate recombination and form joint-molecule intermediates is well understood. To shed light on subsequent events, we exploited the I-SceI homing endonuclease of yeast to make breaks at I-SceI cleavage sites engineered into the chromosome. We show that survival depends on RecA and RecBCD, and that subsequent events can proceed via either of two pathways, one dependent on the RuvABC Holliday junction resolvase and the other on RecG helicase. Both pathways rely on PriA, presumably to facilitate DNA replication. We discuss the possibility that classical Holliday junctions may not be essential intermediates in repair and consider alternative pathways for RecG-dependent separation of joint molecules formed by RecA.

Chromosomes, Bacterial↗

PubMed2004

Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes.

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. DeltaleuB and DeltatrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.

3-Isopropylmalate Dehydrogenase↗

PubMed2004

DNA binding by the meningococcal RdgC protein, associated with pilin antigenic variation.

The RdgC protein of Neisseria gonorrhoeae is required for efficient pilin antigenic variation, although its precise role has yet to be established. We demonstrate that the nearly identical RdgC from Neisseria meningitidis binds DNA with little specificity for sequence or structure, like the Escherichia coli protein. We also show that neither protein is able to constrain torsional tension in relaxed DNA. These data exclude several possible roles for RdgC in pilin antigenic variation and suggest that RdgC performs a similar function in both E. coli and the Neisseria spp.

Amino Acid Sequence↗

PubMed2003

Crystallization of RusA Holliday junction resolvase from Escherichia coli.

Crystals of the Escherichia coli Holliday junction resolvase RusA have been obtained using the hanging-drop method and characterized. The crystals have a primitive monoclinic form and belong to space group P2(1). The V(M) value suggests the presence of two copies of the monomer in the asymmetric unit. A full three-wavelength MAD data collection on a selenomethionine-incorporated form has been undertaken and structure determination is under way using data collected to 2.1 A resolution.

Crystallization↗

PubMed2003

A model for dsDNA translocation revealed by a structural motif common to RecG and Mfd proteins.

RecG protein differs from other helicases analysed to atomic resolution in that it mediates strand separation via translocation on double-stranded (ds) rather than single-stranded (ss) DNA. We describe a highly conserved helical hairpin motif in RecG and show it to be important for helicase activity. It places two arginines (R609 and R630) in opposing positions within the component helices where they are stabilized by a network of hydrogen bonds involving a glutamate from helicase motif VI. We suggest that disruption of this feature, triggered by ATP hydrolysis, moves an adjacent loop structure in the dsDNA-binding channel and that a swinging arm motion of this loop drives translocation. Substitutions that reverse the charge at R609 or R630 reduce DNA unwinding and ATPase activities, and increase dsDNA binding, but do not affect branched DNA binding. Sequences forming the helical hairpin and loop structures are highly conserved in Mfd protein, a transcription-coupled DNA repair factor that also translocates on dsDNA. The possibility of type I restriction enzymes and chromatin-remodelling factors using similar structures to drive translocation on dsDNA is discussed.

Adenosine Triphosphate↗

PubMed2003

The RdgC protein of Escherichia coli binds DNA and counters a toxic effect of RecFOR in strains lacking the replication restart protein PriA.

PriA protein provides a means to load the DnaB replicative helicase at DNA replication fork and D loop structures, and is therefore a key factor in the rescue of stalled or broken forks and subsequent replication restart. We show that the nucleoid-associated RdgC protein binds non-specifically to single-stranded (ss) DNA and double-stranded DNA. It is also essential for growth of a strain lacking PriA, indicating that it might affect replication fork progression or fork rescue. dnaC suppressors of priA overcome this inviability, especially when RecF, RecO or RecR is inactivated, indicating that RdgC avoids or counters a toxic effect of these proteins. Mutations modifying ssDNA-binding (SSB) protein also negate this toxic effect, suggesting that the toxicity reflects inappropriate loading of RecA on SSB-coated ssDNA, leading to excessive or untimely RecA activity. We suggest that binding of RdgC to DNA limits RecA loading, avoiding problems at replication forks that would otherwise require PriA to promote replication restart. Mutations in RNA polymerase also reduce the toxic effect of RecFOR, providing a further link between DNA replication, transcription and repair.

Cell Division↗

PubMed2003

The structure of Escherichia coli RusA endonuclease reveals a new Holliday junction DNA binding fold.

Holliday junction resolution performed by a variety of structure-specific endonucleases is a key step in DNA recombination and repair. It is believed that all resolvases carry out their reaction chemistries in a similar fashion, utilizing a divalent cation to facilitate the hydrolysis of the phosphodiester backbone of the DNA, but their architecture varies. To date, with the exception of bacteriophage T4 endonuclease VII, each of the known resolvase enzyme structures has been categorized into one of two families: the integrases and the nucleases. We have now determined the structure of the Escherichia coli RusA Holliday junction resolvase, which reveals a fourth structural class for these enzymes. The structure suggests that dimer formation is essential for Mg(2+) cation binding and hence catalysis and that like the other resolvases, RusA distorts its Holliday junction target upon binding. Key residues identified by mutagenesis experiments are well positioned to interact with the DNA.

Amino Acid Sequence↗

PubMed2003

PriA supports two distinct pathways for replication restart in UV-irradiated Escherichia coli cells.

The Escherichia coli PriA protein loads the DnaB replicative helicase at branched DNA structures independently of the replication initiator protein, DnaA, and thereby facilitates assembly of functional replisomes at sites removed from oriC. It is therefore a critical factor in the rescue of replication forks stalled at DNA lesions. It is also a DNA helicase. We describe insertions near the 3' end of priA that interfere with PriA activity. These insertions and the previously described priA300 encoding helicase-defective PriA K230R are shown to be effective suppressors of the DNA repair defect in recG strains, but substantially reduce the ability of ruv mutants to survive DNA damage. The data presented suggest that PriA helicase in conjunction with RecG can promote direct rescue of stalled forks independently of the recombinational pathway promoted by the combined activities of the RuvABC, RecBCD and RecA proteins, which requires only the primosome assembly activity of PriA to load DnaB at D loops. In cells lacking the helicase activity of PriA, we propose that stalled forks can be redirected to the recombination pathway via a Holliday junction intermediate common to both pathways, thus explaining the resistance of these cells to DNA damage.

Adenosine Triphosphatases↗

PubMed2002

Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase.

The RuvABC and RecBCD proteins promote rescue of stalled or broken DNA replication forks in Escherichia coli. Strains lacking these proteins cope poorly with DNA damage and have problems with chromosome segregation and cell division. We show how these difficulties are overcome to varying degrees by a sub-class of RNA polymerase mutations selected for their stringent phenotype. Thirty-five mutations were sequenced. All but one change single amino acids in RpoB or RpoC that lie on or near the path taken by DNA through the enzyme, indicating they may affect the stability of transcription complexes. Four mutant enzymes are shown to form unstable open complexes at the lambdacro promoter. At least one may also release stalled complexes or limit their formation, as it reduces the need for reactivation of transcription by GreA or GreB, and for transcription-coupled DNA repair of UV damage by Mfd. The results shed light on the interplay between DNA replication and transcription and suggest ways in which conflicts between these two vital cellular processes are avoided or resolved.

Bacterial Proteins↗

PubMed2002

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.

Bacterial Proteins↗