ORCID Profile
0000-0003-1543-9342
Current Organisation
University of St Andrews
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Publisher: Springer Science and Business Media LLC
Date: 30-04-2008
DOI: 10.1038/NATURE06883
Abstract: Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences erse endpoints in the cellular DNA damage response.
Publisher: Oxford University Press (OUP)
Date: 11-10-2004
DOI: 10.1093/NAR/GKH869
Publisher: CSIRO Publishing
Date: 2014
DOI: 10.1071/CH14342
Abstract: The protein Hjc from the thermophilic archaeon Sulfolobus solfataricus (Ss) presented many challenges to both structure solution and formation of stable complexes with its substrate, the DNA four-way or Holliday junction. As the challenges were caused by an uncharacteristically high propensity for rapid and promiscuous crystallisation, we investigated the molecular cause of this behaviour, corrected it by mutagenesis, and solved the X-ray crystal structures of the two mutants. An active site mutant SsHjcA32A crystallised in space group I23 (a 144.2 Å 68 % solvent), and a deletion of a key crystal contact site, SsHjcδ62–63 crystallised in space group P21 (a 64.60, b 61.83, c 55.25 Å β = 95.74° 28 % solvent). Characterisation and comparative analysis of the structures are presented along with discussion of the pitfalls of the use of protein engineering to alter crystallisability while maintaining biological function.
Publisher: Elsevier BV
Date: 03-2002
Publisher: Portland Press Ltd.
Date: 06-01-2015
DOI: 10.1042/BJ20141140
Abstract: Canonical single-stranded DNA-binding proteins (SSBs) from the oligosaccharide/oligonucleotide-binding (OB) domain family are present in all known organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a ‘simple’ domain organization consisting of a single DNA-binding OB fold coupled to a flexible C-terminal tail, in contrast with other SSBs in this family that incorporate up to four OB domains. Despite the large differences in the domain organization within the SSB family, the structure of the OB domain is remarkably similar all cellular life forms. However, there are significant differences in the molecular mechanism of ssDNA binding. We have determined the structure of the SsoSSB OB domain bound to ssDNA by NMR spectroscopy. We reveal that ssDNA recognition is modulated by base-stacking of three key aromatic residues, in contrast with the OB domains of human RPA and the recently discovered human homologue of SsoSSB, hSSB1. We also demonstrate that SsoSSB binds ssDNA with a footprint of five bases and with a defined binding polarity. These data elucidate the structural basis of DNA binding and shed light on the molecular mechanism by which these ‘simple’ SSBs interact with ssDNA.
Publisher: International Union of Crystallography (IUCr)
Date: 31-10-2008
Publisher: Oxford University Press (OUP)
Date: 13-02-2004
DOI: 10.1093/NAR/GKH259
Publisher: Oxford University Press (OUP)
Date: 08-03-2005
DOI: 10.1093/NAR/GKI288
Publisher: Elsevier BV
Date: 02-2001
Publisher: Oxford University Press (OUP)
Date: 03-11-2010
DOI: 10.1093/NAR/GKQ1098
Publisher: Oxford University Press (OUP)
Date: 10-01-2011
DOI: 10.1093/NAR/GKQ1340
Publisher: Elsevier BV
Date: 2002
Publisher: Springer Science and Business Media LLC
Date: 24-04-2010
Publisher: International Union of Crystallography (IUCr)
Date: 20-12-2002
DOI: 10.1107/S0907444902019546
Abstract: HolliDay junction endonuclease (Hje) from Sulfolobus solfataricus is a resolving enzyme involved in cleaving specific sites on either side of recombinant four-way HolliDay junctions. The HJE gene from S. solfataricus was cloned from genomic DNA into the pET19b Escherichia coli expression vector and recombinant protein was expressed to high levels. Hje was purified using heat treatment, cation exchange and gel filtration. Hanging-drop crystallization trials yielded primitive hexagonal crystals which diffract to 2.4 A on a laboratory source. Systematic absences (only 00l = 6n present) and poor scaling in P622 indicate that the space group is P6(1) or its enantiomer. Failed attempts at molecular replacement using models of a related archaeal resolving enzyme, Hjc, raise the possibility of a difference in quaternary structure between Hjc and Hje, which may be responsible for differences in their activities.
Publisher: Proceedings of the National Academy of Sciences
Date: 05-2001
Abstract: The 2.15-Å structure of Hjc, a Holliday junction-resolving enzyme from the archaeon Sulfolobus solfataricus , reveals extensive structural homology with a superfamily of nucleases that includes type II restriction enzymes. Hjc is a dimer with a large DNA-binding surface consisting of numerous basic residues surrounding the metal-binding residues of the active sites. Residues critical for catalysis, identified on the basis of sequence comparisons and site-directed mutagenesis studies, are clustered to produce two active sites in the dimer, about 29 Å apart, consistent with the requirement for the introduction of paired nicks in opposing strands of the four-way DNA junction substrate. Hjc displays similarity to the restriction endonucleases in the way its specific DNA-cutting pattern is determined but uses a different arrangement of nuclease subunits. Further structural similarity to a broad group of metal hosphate-binding proteins, including conservation of active-site location, is observed. A high degree of conservation of surface electrostatic character is observed between Hjc and T4-phage endonuclease VII despite a complete lack of structural homology. A model of the Hjc–Holliday junction complex is proposed, based on the available functional and structural data.
Publisher: Springer Science and Business Media LLC
Date: 02-2009
DOI: 10.1038/NCHEMBIO.145
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Malcolm F White.