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1.  Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates 
Nucleic Acids Research  2013;42(3):1857-1872.
Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5′ flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5′ ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex.
PMCID: PMC3919604  PMID: 24234453
2.  Structure of the archaeal Cascade subunit Csa5 
RNA Biology  2013;10(5):762-769.
The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.
PMCID: PMC3737334  PMID: 23846216
CRISPR; Csa5; structure; CRISPR interference; Cascade
The Journal of biological chemistry  2007;283(8):5118-5126.
Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest, and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high-resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as a molecular brake, clamping the ssDNA extruded through the central pore of the helicase structure to limit the enzyme’s helicase activity. This provides an elegant mechanism to tune the enzyme’s processivity to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, suggesting that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates.
PMCID: PMC3434800  PMID: 18056710
4.  Structure of the DNA repair helicase XPD 
Cell  2008;133(5):801-812.
The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalysing DNA duplex opening localised to the transcription start site or site of DNA damage, respectively. XPD has a 5′ to 3′ polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigentosum, trichothiodystrophy and Cockayne’s syndrome, characterised by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 Å crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.
PMCID: PMC3326533  PMID: 18510925
5.  Extensive Lysine Methylation in Hyperthermophilic Crenarchaea: Potential Implications for Protein Stability and Recombinant Enzymes 
Archaea  2010;2010:106341.
In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in α-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.
PMCID: PMC2929605  PMID: 20811616
6.  PCNA and XPF cooperate to distort DNA substrates 
Nucleic Acids Research  2009;38(5):1664-1675.
XPF is a structure-specific endonuclease that preferentially cleaves 3′ DNA flaps during a variety of repair processes. The crystal structure of a crenarchaeal XPF protein bound to a DNA duplex yielded insights into how XPF might recognise branched DNA structures, and recent kinetic data have demonstrated that the sliding clamp PCNA acts as an essential cofactor, possibly by allowing XPF to distort the DNA structure into a proper conformation for efficient cleavage to occur. Here, we investigate the solution structure of the 3′-flap substrate bound to XPF in the presence and absence of PCNA using intramolecular Förster resonance energy transfer (FRET). We demonstrate that recognition of the flap substrate by XPF involves major conformational changes of the DNA, including a 90° kink of the DNA duplex and organization of the single-stranded flap. In the presence of PCNA, there is a further substantial reorganization of the flap substrate bound to XPF, providing a structural basis for the observation that PCNA has an essential catalytic role in this system. The wider implications of these observations for the plethora of PCNA-dependent enzymes are discussed.
PMCID: PMC2836553  PMID: 20008103
7.  Protein-Protein Interactions Leading to Recruitment of the Host DNA Sliding Clamp by the Hyperthermophilic Sulfolobus islandicus Rod-Shaped Virus 2 
Journal of Virology  2014;88(12):7105-7108.
Viruses infecting hyperthermophilic archaea typically do not encode DNA polymerases, raising questions regarding their genome replication. Here, using a yeast two-hybrid approach, we have assessed interactions between proteins of Sulfolobus islandicus rod-shaped virus 2 (SIRV2) and the host-encoded proliferating cell nuclear antigen (PCNA), a key DNA replication protein in archaea. Five SIRV2 proteins were found to interact with PCNA, providing insights into the recruitment of host replisome for viral DNA replication.
PMCID: PMC4054388  PMID: 24696494
8.  Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system 
Nucleic Acids Research  2014;42(10):6532-6541.
CRISPR-Cas is an adaptive prokaryotic immune system, providing protection against viruses and other mobile genetic elements. In type I and type III CRISPR-Cas systems, CRISPR RNA (crRNA) is generated by cleavage of a primary transcript by the Cas6 endonuclease and loaded into multisubunit surveillance/effector complexes, allowing homology-directed detection and cleavage of invading elements. Highly studied CRISPR-Cas systems such as those in Escherichia coli and Pseudomonas aeruginosa have a single Cas6 enzyme that is an integral subunit of the surveillance complex. By contrast, Sulfolobus solfataricus has a complex CRISPR-Cas system with three types of surveillance complexes (Cascade/type I-A, CSM/type III-A and CMR/type III-B), five Cas6 paralogues and two different CRISPR-repeat families (AB and CD). Here, we investigate the kinetic properties of two different Cas6 paralogues from S. solfataricus. The Cas6-1 subtype is specific for CD-family CRISPR repeats, generating crRNA by multiple turnover catalysis whilst Cas6-3 has a broader specificity and also processes a non-coding RNA with a CRISPR repeat-related sequence. Deep sequencing of crRNA in surveillance complexes reveals a biased distribution of spacers derived from AB and CD loci, suggesting functional coupling between Cas6 paralogues and their downstream effector complexes.
PMCID: PMC4041471  PMID: 24753403
9.  CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus 
Nucleic Acids Research  2014;42(8):5280-5288.
The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene (β-galactosidase) using an artificial miniCRISPR locus. With a single complementary spacer, ∼50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs (i.e. analogous to RNA interference in eukaryotes) and the re-programming of the system to silence specific genes of interest.
PMCID: PMC4005642  PMID: 24603867
10.  Structure of the CRISPR Interference Complex CSM Reveals Key Similarities with Cascade 
Molecular Cell  2013;52(1):124-134.
The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I–III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family.
Graphical Abstract
•The CSM complex from Sulfolobus solfataricus has been purified and characterized•EM reveals a helical backbone with striking similarities to the Cascade complex•Mass spectrometry defines the subunit stoichiometry and organization of the complex•CSM subunits are modified by methylation, acetylation, and phosphorylation
PMCID: PMC3807668  PMID: 24119402
11.  CRISPR interference: a structural perspective 
Biochemical Journal  2013;453(Pt 2):155-166.
CRISPR (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. Genomically encoded crRNA (CRISPR RNA) is used by Cas (CRISPR-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. The process is known as ‘interference’. In the present review we cover recent progress on the structural biology of the CRISPR/Cas system, focusing on the Cas proteins and complexes that catalyse crRNA biogenesis and interference. Structural studies have helped in the elucidation of key mechanisms, including the recognition and cleavage of crRNA by the Cas6 and Cas5 proteins, where remarkable diversity at the level of both substrate recognition and catalysis has become apparent. The RNA-binding RAMP (repeat-associated mysterious protein) domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10. Structural analysis has also revealed an evolutionary link between the small subunits of the type I and type III-B interference complexes. Future studies of the interference complexes and their constituent components will transform our understanding of the system.
PMCID: PMC3727216  PMID: 23805973
antiviral defence; cluster of regularly interspaced palindromic repeats (CRISPR); crystallography; evolution; protein structure; repeat-associated mysterious protein (RAMP); BhCas5c, Bacillus halodurans Cas5c; CRISPR, cluster of regularly interspaced palindromic repeats; Cas, CRISPR-associated; Cascade, CRISPR-associated complex for antiviral defence; crRNA, CRISPR RNA; dsDNA, double-stranded DNA; EcoCas3, Escherichia coli Cas3; EM, electron microscopy; HD, histidine–aspartate; MjaCas3″, Methanocaldococcus jannaschii Cas3″; PaCas6f, Pseudomonas aeruginosa Cas6f; PAM, protospacer adjacent motif; PfuCas, Pyrococcus furiosus Cas; pre-crRNA, precursor crRNA; RAMP, repeat-associated mysterious protein; RRM, RNA recognition motif; ssDNA, single-stranded DNA; SsoCas, Sulfolobus solfataricus Cas; ssRNA, single-stranded RNA; SthCas3, Streptococcus thermophilus Cas3; tracrRNA, trans-activating crRNA; TtCas, Thermus thermophilus Cas
12.  Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing 
Biochemical Journal  2013;452(Pt 2):223-230.
The competition between viruses and hosts is played out in all branches of life. Many prokaryotes have an adaptive immune system termed ‘CRISPR’ (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral DNA. The captured DNA is integrated into the genomic DNA of the organism flanked by direct repeats, transcribed and processed to generate crRNA (CRISPR RNA) that is loaded into a variety of effector complexes. These complexes carry out sequence-specific detection and destruction of invading mobile genetic elements. In the present paper, we report the structure and activity of a Cas6 (CRISPR-associated 6) enzyme (Sso1437) from Sulfolobus solfataricus responsible for the generation of unit-length crRNA species. The crystal structure reveals an unusual dimeric organization that is important for the enzyme's activity. In addition, the active site lacks the canonical catalytic histidine residue that has been viewed as an essential feature of the Cas6 family. Although several residues contribute towards catalysis, none is absolutely essential. Coupled with the very low catalytic rate constants of the Cas6 family and the plasticity of the active site, this suggests that the crRNA recognition and chaperone-like activities of the Cas6 family should be considered as equal to or even more important than their role as traditional enzymes.
PMCID: PMC3652601  PMID: 23527601
antiviral defence; Cas6; clustered regularly interspaced short palindromic repeats (CRISPR); ribonuclease; Sulfolobus; CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR-associated; crRNA, CRISPR RNA; Ni-NTA, Ni2+-nitrilotriacetate; PaCas6f, Pseudomonas aeruginosa Cas6; PfuCas6, Pyrococcus furiosus Cas6; RAMP, repeat-associated mysterious protein; RMSD, root mean square deviation; RRM, RNA-recognition motif; SAD, single-wavelength anomalous dispersion; SsoCas6, Sulfolobus solfataricus Cas6; TBE, Tris/borate/EDTA; TEV, tobacco etch virus; TtCas6e, Thermus thermophilus Cas6
13.  Crenarchaeal chromatin proteins Cren7 and Sul7 compact DNA by inducing rigid bends 
Nucleic Acids Research  2012;41(1):196-205.
Archaeal chromatin proteins share molecular and functional similarities with both bacterial and eukaryotic chromatin proteins. These proteins play an important role in functionally organizing the genomic DNA into a compact nucleoid. Cren7 and Sul7 are two crenarchaeal nucleoid-associated proteins, which are structurally homologous, but not conserved at the sequence level. Co-crystal structures have shown that these two proteins induce a sharp bend on binding to DNA. In this study, we have investigated the architectural properties of these proteins using atomic force microscopy, molecular dynamics simulations and magnetic tweezers. We demonstrate that Cren7 and Sul7 both compact DNA molecules to a similar extent. Using a theoretical model, we quantify the number of individual proteins bound to the DNA as a function of protein concentration and show that forces up to 3.5 pN do not affect this binding. Moreover, we investigate the flexibility of the bending angle induced by Cren7 and Sul7 and show that the protein–DNA complexes differ in flexibility from analogous bacterial and eukaryotic DNA-bending proteins.
PMCID: PMC3592393  PMID: 23155062
14.  The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster 
PLoS ONE  2012;7(10):e47232.
The Cas4 protein is one of the core CRISPR-associated (Cas) proteins implicated in the prokaryotic CRISPR system for antiviral defence. Cas4 is thought to play a role in the capture of new viral DNA sequences for incorporation into the host genome. No biochemical activity has been reported for Cas4, but it is predicted to include a RecB nuclease domain. We show here that Cas4 family proteins from the archaeon Sulfolobus solfataricus utilise four conserved cysteine residues to bind an iron-sulfur cluster in an arrangement reminiscent of the AddB nuclease of Bacillus subtilis. The Cas4 family protein Sso0001 is a 5′ to 3′ single stranded DNA exonuclease in vitro that is stalled by extrahelical DNA adducts. A role for Cas4 in DNA duplex strand resectioning to generate recombinogenic 3′ single stranded DNA overhangs is proposed. Comparison of the AddB structure with that of a related bacterial nuclease from Eubacterium rectales reveals that the iron-sulfur cluster can be replaced by a zinc ion without disrupting the protein structure, with implications for the evolution of iron-sulfur binding proteins.
PMCID: PMC3466216  PMID: 23056615
15.  Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR 
Nucleic Acids Research  2012;41(1):e11.
Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.
PMCID: PMC3592447  PMID: 22941643
16.  Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity 
Molecular cell  2012;45(3):303-313.
The prokaryotic Clusters of Regularly Interspaced Palindromic Repeats (CRISPR) system utilizes genomically-encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse “payload” of targeting crRNA. The crystal structure of Cmr7 and low resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8-nucleotide repeat-derived 5′ sequence in the crRNA, but not on the presence of a proto-spacer associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.
PMCID: PMC3381847  PMID: 22227115
17.  Structural and functional characterisation of a conserved archaeal RadA paralog with antirecombinase activity 
Journal of molecular biology  2009;389(4):661-673.
DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand-exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. In bacteria, RecA is the sole protein responsible for this reaction, whereas, in eukaryotes, there are several RAD51 paralogs that cooperate to catalyse strand exchange. All archaea have at least one (and as many as four) RadA paralogs, but their function remains unclear. Here we show the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions, and are not UV-inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large aRadC sub-family of archaeal RadA paralogs, functions as an ATPase that binds tightly to ssDNA. However, Sso2452 is not an active recombinase in vitro, and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.
PMCID: PMC3387904  PMID: 19414020
Archaea; Recombinase; RadA; Homologous Recombination; Strand Exchange
18.  An unusual chromophore and novel crosslinks in ranasmurfin - a blue protein from the foam nests of a tropical frog 
Ranasmurfin is an unusual blue protein isolated from the nests of a Malaysian tree frog, Polypedates leucomystax,[1] showing the rich chemical diversity displayed by biomolecular foams. Many species of tropical frogs use foams to protect delicate eggs and developing embryos against environmental challenges. These nests act as miniature ecosystems containing a spectrum of novel proteins and other macromolecules with functions related to foam stabilization and adhesion, resistance to microbial degradation, predation, or dehydration, providing a biocompatible environment for embryonic development.Thisworkformspartofourwiderstudyofthe intriguing physical and chemical properties of biofoams as unusual examples of biological soft matter.[2]
PMCID: PMC3313644  PMID: 18781570
19.  Staphylococcus aureus DinG, a helicase that has evolved into a nuclease 
Biochemical Journal  2012;442(Pt 1):77-84.
DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.
PMCID: PMC3270479  PMID: 22166102
damage inducible gene G (DinG); DNA repair; helicase; iron–sulfur; nuclease; xeroderma pigmentosum complementation group D (XPD); CRISPR, clustered regularly interspaced short palindromic repeats; DinG, damage inducible gene G; DTT, dithiothreitol; FAM, 6-carboxyfluorescein; FeS, iron–sulfur; sarDinG, Staphylococcus aureus DinG; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA; TBE, Tris/borate/EDTA; TEV, tobacco etch virus; XPD, xeroderma pigmentosum complementation group D; XPF, xeroderma pigmentosum complementation group F; WT, wild-type
20.  A Dimeric Rep Protein Initiates Replication of a Linear Archaeal Virus Genome: Implications for the Rep Mechanism and Viral Replication ▿ †  
Journal of Virology  2010;85(2):925-931.
The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between an active-site tyrosine and the 5′ end of the DNA, releasing a 3′ DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral replication are discussed.
PMCID: PMC3019997  PMID: 21068244
21.  hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity 
Nucleic Acids Research  2011;39(9):3643-3651.
hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.
PMCID: PMC3089470  PMID: 21227926
22.  hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex 
Nucleic Acids Research  2010;39(5):1692-1702.
hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).
PMCID: PMC3061066  PMID: 21051358
23.  Purification, crystallization and data collection of Pectobacterium chrysanthemi AcsD, a type A siderophore synthetase 
AcsD, an NRPS-independent siderophore synthetase, has been crystallized.
AcsD, a type A siderophore synthetase with a molecular weight of 71 140 Da from Pectobacterium chrysanthemi, has been expressed, purified and crystallized at 293 K. The protein crystallized in the primitive orthorhombic space group P212121, with unit-cell parameters a = 80.3, b = 95.7, c = 161.1 Å, α = β = γ = 90°. Systematic absences were consistent with space group P212121. A complete data set has been collected to 2.25 Å resolution on BM14 at the ESRF. Consideration of the likely solvent content suggested that the asymmetric unit contained two molecules. Gel-filtration experiments indicated that the protein was a dimer, although self-rotation analyses did not detect a convincing twofold symmetry axis in the asymmetric unit. The protein has no convincing sequence match to any known structure and thus solution is likely to require experimental phasing.
PMCID: PMC2581690  PMID: 18997340
siderophore synthetases; Pectobacterium chrysanthemi
24.  The Scottish Structural Proteomics Facility: targets, methods and outputs 
The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.
Electronic supplementary material
The online version of this article (doi:10.1007/s10969-010-9090-y) contains supplementary material, which is available to authorized users.
PMCID: PMC2883930  PMID: 20419351
High-throughput; Protein crystallography; Structural proteomics; SSPF
25.  Dimer–dimer stacking interactions are important for nucleic acid binding by the archaeal chromatin protein Alba 
Biochemical Journal  2010;427(Pt 1):49-55.
Archaea use a variety of small basic proteins to package their DNA. One of the most widespread and highly conserved is the Alba (Sso10b) protein. Alba interacts with both DNA and RNA in vitro, and we show in the present study that it binds more tightly to dsDNA (double-stranded DNA) than to either ssDNA (single-stranded DNA) or RNA. The Alba protein is dimeric in solution, and forms distinct ordered complexes with DNA that have been visualized by electron microscopy studies; these studies suggest that, on binding dsDNA, the protein forms extended helical protein fibres. An end-to-end association of consecutive Alba dimers is suggested by the presence of a dimer–dimer interface in crystal structures of Alba from several species, and by the strong conservation of the interface residues, centred on Arg59 and Phe60. In the present study we map perturbation of the polypeptide backbone of Alba upon binding to DNA and RNA by NMR, and demonstrate the central role of Phe60 in forming the dimer–dimer interface. Site-directed spin labelling and pulsed ESR are used to confirm that an end-to-end, dimer–dimer interaction forms in the presence of dsDNA.
PMCID: PMC2841500  PMID: 20082605
Alba; archaea; ESR; NMR; site-directed spin labelling; cw-ESR, continuous wave ESR; DEER, double electron–electron resonance; dsDNA, double-stranded DNA; EMSA, electrophoretic mobility-shift assay; FRET, fluorescence resonance energy transfer; HSQC, heteronuclear single-quantum coherence; SDSL, site-directed spin labelling; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA

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