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Dale B Wigley

Publications and source records attributed to Dale B Wigley.

14 recordsLinked to original sources
PubMed2006

Biochemical analysis of a DNA replication origin in the archaeon Aeropyrum pernix.

We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple origin recognition box (ORB) sites that we show to be a replication origin. At this origin, there are four ORB elements disposed either side of an A+T-rich region. An ORC1 protein dimer binds at each of these ORB sites. Once all four ORB sites have bound ORC1 protein, there is a transition to a higher-order assembly with a defined alteration in topology and superhelicity. Furthermore, after this transition, the A+T-rich region becomes sensitive to digestion by DNase I and P1 nuclease, revealing that the transition promotes distortion of the DNA in this region, presumably as a prelude to loading of MCM helicase.

Aeropyrum↗
PubMed2006

CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing.

The BRCA2 tumour suppressor regulates the RAD-51 recombinase during double-strand break (DSB) repair by homologous recombination (HR) but how BRCA2 executes its functions is not well understood. We previously described a functional homologue of BRCA2 in Caenorhabditis elegans (CeBRC-2) that binds preferentially to single-stranded DNA via an OB-fold domain and associates directly with RAD-51 via a single BRC domain. Consistent with a direct role in HR, Cebrc-2 mutants are defective for repair of meiotic and radiation-induced DSBs due to an inability to regulate RAD-51. Here, we explore the function of CeBRC-2 in HR processes using purified proteins. We show that CeBRC-2 stimulates RAD-51-mediated D-loop formation and reduces the rate of ATP hydrolysis catalysed by RAD-51. These functions of CeBRC-2 are dependent upon direct association with RAD-51 via its BRC motif and on its DNA-binding activity, as point mutations in the BRC domain that abolish RAD-51 binding or the BRC domain of CeBRC-2 alone, lacking the DNA-binding domain, fail to stimulate RAD-51-mediated D-loop formation and do not reduce the rate of ATP hydrolysis by RAD-51. Phenotypic comparison of Cebrc-2 and rad-51 mutants also revealed a role for CeBRC-2 in an error-prone DSB repair pathway independent of rad-51 and non-homologous end joining, raising the possibility that CeBRC-2 may have replaced the role of vertebrate Rad52 in DNA single-strand annealing (SSA), which is missing from C. elegans. Indeed, we show here that CeBRC-2 mediates SSA of RPA-oligonucleotide complexes similar to Rad52. These results reveal RAD-51-dependent and -independent functions of CeBRC-2 that provide an explanation for the difference in DNA repair defects observed in Cebrc-2 and rad-51 mutants, and define mechanistic roles for CeBRC-2 in HR and in the SSA pathway for DSB repair.

Adenosine Triphosphate↗
PubMed2006

Communication between subunits within an archaeal clamp-loader complex.

We have investigated the communication between subunits in replication factor C (RFC) from Archaeoglobus fulgidus. Mutation of the proposed arginine finger in the small subunits results in a complex that can still bind ATP but has impaired clamp-loading activity, a process that normally only requires binding of nucleotide. The small subunit alone forms a hexameric ring that is six-fold symmetric in the absence of ATP. However, this symmetry is broken when the nucleotide is bound to the complex. A conformational change associated with nucleotide binding may relate to the opening of PCNA rings by RFC during the loading reaction. The structures also reveal the importance of the N-terminal helix of each subunit at the ATP-binding site. Analysis of mutant protein complexes containing subunits lacking this N-terminal helix reveals key distinct regulatory roles during clamp loading that are different for the large and small subunits in the RFC complex.

Adenosine Triphosphate↗
PubMed2005

Pumps, paradoxes and ploughshares: mechanism of the MCM2-7 DNA helicase.

In eukaryotes, numerous lines of evidence have coalesced into a convincing case that the MCM2-7 complex - a heterohexameric ATPase - is the replicative DNA helicase. However, almost nothing is known about how this enzyme functions in a cellular context. Some models for the mechanism of the MCM2-7 helicase envision that it translocates along single-stranded DNA (ssDNA), whereas, more recently, it is has been suggested that it pumps double-stranded DNA (dsDNA) through its central channel. In particular, one model in which a double hexamer of MCM2-7 pumps dsDNA towards the hexamer interface and extrudes ssDNA laterally as a result of torsional strain is gaining popularity. Here, we discuss existing models and propose a new variation in which a single hexamer is the functional unit of the helicase. Duplex DNA is pumped into MCM2-7 and, as it emerges from the complex, a rigid protein that we term the 'ploughshare' splits the duplex.

Animals↗
PubMed2004

Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks.

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.

Crystallization↗
PubMed2004

Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix.

Archaea contain one or more proteins with homology to eukaryotic ORC/Cdc6 proteins. Sequence analysis suggests the existence of at least two subfamilies of these proteins, for which we propose the nomenclature ORC1 and ORC2. We have determined crystal structures of the ORC2 protein from the archaeon Aeropyrum pernix in complexes with ADP or a non-hydrolysable ATP analogue, ADPNP. Between two crystal forms, there are three crystallographically independent views of the ADP complex and two of the ADPNP complex. The protein molecules in the three complexes with ADP adopt very different conformations, while the two complexes with ADPNP are the same. These structures indicate that there is considerable conformational flexibility in ORC2 but that ATP binding stabilises a single conformation. We show that the ORC2 protein can bind DNA, and that this activity is associated with the C-terminal domain of the protein. We present a model for the interaction of the winged helix (WH) domain of ORC2 with DNA that differs from that proposed previously for Pyrobaculum aerophilum ORC/Cdc6.

Adenosine Diphosphate↗
PubMed2004

Distinct roles for ATP binding and hydrolysis at individual subunits of an archaeal clamp loader.

Circular clamps are utilised by replicative polymerases to enhance processivity. The topological problem of loading a toroidal clamp onto DNA is overcome by ATP-dependent clamp loader complexes. Different organisms use related protein machines to load clamps, but the mechanisms by which they utilise ATP are surprisingly different. Using mutant clamp loaders that are deficient in either ATP binding or hydrolysis in different subunits, we show how the different subunits of an archaeal clamp loader use ATP binding and hydrolysis in distinct ways at different steps in the loading process. Binding of nucleotide by the large subunit and three of the four small subunits is sufficient for clamp loading. However, ATP hydrolysis by the small subunits is required for release of PCNA to allow formation of the complex between PCNA and the polymerase, while hydrolysis by the large subunit is required for catalytic clamp loading.

Adenosine Triphosphatases↗
PubMed2003

Multiple roles for ATP hydrolysis in nucleic acid modifying enzymes.

Enzymes that operate on nucleic acid substrates are faced with the unusual situation where the substrate is much larger than themselves. Despite the potential to promote catalysis by utilizing the significant binding energy available through their interaction with substrate, ATP hydrolysis is frequently a part of the mechanism of these enzymes. The reasons for this have become clearer in recent years, and a surprising range of ways that these enzymes utilize the free energy of hydrolysis of ATP has been revealed. This review describes these different mechanisms in the context of the biochemical reactions that they support.

Adenosine Triphosphate↗
PubMed2003

Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus.

The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing. Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea. The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue. The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro. The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA. Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA. The purified Orc/Cdc6 homologue was also able to bind DNA. Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates. Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap. The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer.

Adenosine Triphosphatases↗
PubMed2002

Biochemical characterisation of the clamp/clamp loader proteins from the euryarchaeon Archaeoglobus fulgidus.

Replicative polymerases of eukaryotes, prokaryotes and archaea obtain processivity using ring-shaped DNA sliding clamps that are loaded onto DNA by clamp loaders [replication factor C (RFC) in eukaryotes]. In this study, we cloned the two genes for the subunits of the RFC homologue of the euryarchaeon Archaeoglobus fulgidus. The proteins were expressed and purified from Escherichia coli both individually and as a complex. The afRFC subunits form a heteropentameric complex consisting of one copy of the large subunit and four copies of the small subunits. To analyse the functionality of afRFC, we also expressed the A.fulgidus PCNA homologue and a type B polymerase (PolB1) in E.coli. In primer extension assays, afRFC stimulated the processivity of afPolB1 in afPCNA-dependent reactions. Although the afRFC complex showed significant DNA-dependent ATPase activity, which could be further stimulated by afPCNA, neither of the isolated afRFC subunits showed this activity. However, both the large and small afRFC subunits showed interaction with afPCNA. Furthermore, we demonstrate that ATP binding, but not hydrolysis, is needed to stimulate interactions of the afRFC complex with afPCNA and DNA.

Adenosine Triphosphatases↗
PubMed2002

Structure of the single-strand annealing domain of human RAD52 protein.

In eukaryotic cells, RAD52 protein plays a central role in genetic recombination and DNA repair by (i) promoting the annealing of complementary single-stranded DNA and (ii) stimulation of the RAD51 recombinase. The single-strand annealing domain resides in the N-terminal region of the protein and is highly conserved, whereas the nonconserved RAD51-interaction domain is located in the C-terminal region. An N-terminal fragment of human RAD52 (residues 1-209) has been purified to homogeneity and, similar to the full-size protein (residues 1-418), shown to promote single-strand annealing in vitro. We have determined the crystal structure of this single-strand annealing domain at 2.7 A. The structure reveals an undecameric (11) subunit ring with extensive subunit contacts. A large, positively charged groove runs along the surface of the ring, readily suggesting a mechanism by which RAD52 presents the single strand for reannealing with complementary single-stranded DNA.

Binding Sites↗
PubMed2002

Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator.

The PriA protein was identified in Escherichia coli as a factor involved in the replication of extrachromosomal elements such as bacteriophage phiX174 and plasmid pBR322. Recent data show that PriA plays an important role in chromosomal replication, by promoting reassembly of the replication machinery during reinitiation of inactivated forks. A gene encoding a product 32% identical to the E.coli PriA protein has been identified in Bacillus subtilis. To characterise this protein, designated PriA(Bs), we constructed priA(Bs) mutants. These mutants are poorly viable, filamentous and sensitive to rich medium and UV irradiation. Replication of pAMbeta1-type plasmids, which is initiated through the formation of a D-loop structure, and the activity of the primosome assembly site ssiA of plasmid pAMbeta1 are strongly affected in the mutants. The purified PriA(Bs) protein binds preferentially to the active strand of ssiA, even in the presence of B.subtilis SSB protein (SSB(Bs)). PriA(Bs) also binds stably and specifically to an artificial D-loop structure in vitro. These data show that PriA(Bs) recognises two specific substrates, ssiA and D-loops, and suggest that it triggers primosome assembly on them. PriA(Bs) also displays a single-stranded DNA-dependent ATPase activity, which is reduced in the presence of SSB(Bs), unless the ssiA sequence is present on the ssDNA substrate. Finally, PriA(Bs) is shown to be an active helicase. Altogether, these results demonstrate a clear functional identity between PriA(Ec) and PriA(Bs). However, PriA(Bs) does not complement an E.coli priA null mutant strain. This host specificity may be due to the divergence between the proteins composing the E.coli and B.subtilis PriA-dependent primosomes.

Adenosine Triphosphatases↗
PubMed2002

Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine.

Use of the fluorescent base analogue 2-aminopurine has provided a direct demonstration of the translocation of PcrA helicase toward the 5'-end of single-stranded DNA. Single 2-aminopurine bases are introduced into otherwise standard oligonucleotides and produce a fluorescence signal when PcrA reaches their position. We demonstrate that random binding of PcrA to ssDNA is followed by translocation in an ATP-dependent manner toward the 5'-terminus at 80 bases per second at 20 degrees C. The data also provide information on the kinetics of ssDNA binding to the helicase and of the protein dissociation from the 5'-end of ssDNA. A full kinetic model is presented for ATP-dependent DNA translocation by PcrA helicase.

2-Aminopurine↗