199,572 research outputs found
Characterisation of a Holliday junction-resolving enzyme from Schizosaccharomyces pombe
No full textWe thank the Cancer Research Campaign for financial support.The rearrangement and repair of DNA by homologous recombination involves the creation of Holliday junctions, which are cleaved by a class of junction-specific endonucleases to generate recombinant duplex DNA products, Only two cellular junction-resolving enzymes have been identified to date: RuvC in eubacteria and CCE1 from Saccharomyces cerevisiae mitochondria, We have identified a protein from Schizosaccharomyces pombe which has 28% sequence identity to CCE1. The YDC2 protein has been, cloned and overexpressed in Escherichia coli, and the purified recombinant protein has been shown to be a Holliday junction-resolving enzyme. YDC2 has a high degree of specificity for the structure of the four-way junction, to which it binds as a dimer, The enzyme exhibits a sequence specificity for junction cleavage that differs from both CCE1 and RuvC, and it cleaves fixed junctions at the point of strand exchange, The conservation of the mechanism of Holliday junction cleavage between two organisms as diverse as S. cerevisiae and S. pombe suggests that there may be a common pathway for mitochondrial homologous recombination in fungi, plants, protists, and possibly higher eukaryotes.Peer reviewe
ruvA Mutants that resolve Holliday junctions but do not reverse replication forks
No full textRuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination
FAN1 activity on asymmetric repair intermediates is mediated by an atypical monomeric virus-type replication-repair nuclease domain
No full textFAN1 is a structure-selective DNA repair nuclease with 5' flap endonuclease activity, involved in the repair of interstrand DNA crosslinks. It is the only eukaryotic protein with a virus-type replication-repair nuclease ("VRR-Nuc") "module" that commonly occurs as a standalone domain in many bacteria and viruses. Crystal structures of three representatives show that they structurally resemble Holliday junction resolvases (HJRs), are dimeric in solution, and are able to cleave symmetric four-way junctions. In contrast, FAN1 orthologs are monomeric and cleave 5' flap structures in vitro, but not Holliday junctions. Modeling of the VRR-Nuc domain of FAN1 reveals that it has an insertion, which packs against the dimerization interface observed in the structures of the viral/bacterial VRR-Nuc proteins. We propose that these additional structural elements in FAN1 prevent dimerization and bias specificity toward flap structures
State of the eastern North Atlantic subpolar gyre: The Extended Ellett Line Programme Annual Report No. 2
No full textThe Extended Ellett Line is a hydrographic section between Iceland and Scotland that is occupied annually by scientists from the National Oceanography Centre (NOC) and the Scottish Association for Marine Science (SAMS), UK. The measurement programme began as a seasonally-occupied hydrographic section in the Rockall Trough in 1975, building on early surface observations made underway from ocean weather ships. In 1996 the section was extended to Iceland, sampling three basins: the Rockall Trough, the Hatton-Rockall Basin and the Iceland Basin. This report presents a summary of data from the Extended Ellett Line programme as well as an overview of activities and analysis from the programme. Historical and recent physical data are analysed to calculate time series of temperature and salinity of the upper ocean and Labrador Sea Water in the eastern subpolar gyre. The most recent Extended Ellett Line cruise (May 2013) is shown as temperature, salinity and density sections
Personal Papers (MS 80-0002)
No full textLetter from Arthur M. Alpert to E. S. Holliday discussing an upcoming meeting of the Industrial Development Committee
Resolution of Joint Molecules by RuvABC and RecG Following Cleavage of the Escherichia coli Chromosome by EcoKI
No full textDNA double-strand breaks can be repaired by homologous recombination involving the formation and resolution of Holliday junctions. In Escherichia coli, the RuvABC resolvasome and the RecG branch-migration enzyme have been proposed to act in alternative pathways for the resolution of Holliday junctions. Here, we have studied the requirements for RuvABC and RecG in DNA double-strand break repair after cleavage of the E. coli chromosome by the EcoKI restriction enzyme. We show an asymmetry in the ability of RuvABC and RecG to deal with joint molecules in vivo. We detect linear DNA products compatible with the cleavage-ligation of Holliday junctions by the RuvABC pathway but not by the RecG pathway. Nevertheless we show that the XerCD-mediated pathway of chromosome dimer resolution is required for survival regardless of whether the RuvABC or the RecG pathway is active, suggesting that crossing-over is a common outcome irrespective of the pathway utilised. This poses a problem. How can cells resolve joint molecules, such as Holliday junctions, to generate crossover products without cleavage-ligation? We suggest that the mechanism of bacterial DNA replication provides an answer to this question and that RecG can facilitate replication through Holliday junctions.</p
[Rev. C. A. Holliday Giving Envelope to Dorothy M. Williams]
No full textPhotograph of Rev. C. A. Holliday, standing at left, handing an envelope to Dorothy M. Williams, at center. Mrs. C. A. Holliday smiles and looks on from the left. Williams is dressed with a dark-colored neck wrap and a white robe
Cellular location and activity of Escherichia coli RecG proteins shed light on the function of its structurally unresolved C-terminus
No full textRecG is a DNA translocase encoded by most species of bacteria. The Escherichia coli protein targets branched DNA substrates and drives the unwinding and rewinding of DNA strands. Its ability to remodel replication forks and to genetically interact with PriA protein have led to the idea that it plays an important role in securing faithful genome duplication. Here we report that RecG co-localises with sites of DNA replication and identify conserved arginine and tryptophan residues near its C-terminus that are needed for this localisation. We establish that the extreme C-terminus, which is not resolved in the crystal structure, is vital for DNA unwinding but not for DNA binding. Substituting an alanine for a highly conserved tyrosine near the very end results in a substantial reduction in the ability to unwind replication fork and Holliday junction structures but has no effect on substrate affinity. Deleting or substituting the terminal alanine causes an even greater reduction in unwinding activity, which is somewhat surprising as this residue is not uniformly present in closely related RecG proteins. More significantly, the extreme C-terminal mutations have little effect on localisation. Mutations that do prevent localisation result in only a slight reduction in the capacity for DNA repair. © 2014 The Author(s)
GEN1 from a thermophilic fungus is functionally closely similar to non-eukaryotic junction-resolving enzymes
No full textProcessing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum, and expressed the N-terminal 487 amino acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1 nucleotide 3' to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme, but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1
Arthur L. Holliday and Eva M. Dreka's wedding invitation
No full textAn invitation and envelope sent to Helen Start of Smyrna, Delaware, to attend the wedding Arthur L. Holliday and Eva M. Dreka on October 12, 1912, in Plattsburg, New York
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