Iain Duggin is an ARC Future Fellow (2017-2020) and leads the Microbial Cell Shape and Structural Dynamics group located at the ithree (infection, immunology and innovation) institute, University of Technology Sydney.
Much of Duggin’s early research focused on microbial chromosome replication and the cell cycle. He developed the first method for archaeal cell cycle synchronization and innovative approaches in functional genomics to establish an understanding of how chromosomes segregate prior to cell division in archaea. More recently, he pioneered a new area of research in the field of prokaryotic cell form and function. In this area he has made breakthrough contributions, including the molecular basis for cell shape changes in halophilic archaea and establishment of a model system for developmental biology of archaea. Archaea is a relatively poorly understood branch of the tree of life that has great potential in biotechnology.
Duggin was awarded a PhD in Molecular Biology from the University of Sydney in 2002. He pursued postdoctoral research at the University of Sydney (2002-2004) before being awarded a MRC Career Development Fellowship that he took up first at the MRC Cancer Cell Unit, Cambridge, UK and then at the Sir William Dunn School of Pathology, University of Oxford, (2005-2009). Following postdoctoral studies at the MRC Laboratory of Molecular Biology (LMB), Cambridge UK (2009-2011), Duggin was recruited to the ithree institute to start his own research group (2011-).
Member of the Australian Society for Microbiology
Member of the Australian Society for Biochemistry and Molecular Biology
Can supervise: YES
Our research focuses on understanding prokaryotic cell dynamics, including prokaryotic morphogenesis, cytoskeleton and the cell cycle. Our aim is to understand gene and protein function at the molecular level, and how these function to effect changes in microbial cell structure and shape in response to environmental conditions. Microbial cells are impressively dynamic, and quick to respond to environmental changes. We are particularly interested in the functions of tubulin-like proteins that control cell morphology and division in archaea (Haloferax volcanii, found in salt lakes) and bacteria (uropathogenic Escherichia coli, found in urinary tract infections), and cytoskeletal proteins in Helicobacter pylori (found in stomach inflammation, ulcers and cancer). Understanding of prokaryotic cell dynamics can spark innovative ideas in the medical and biotechnology sectors.
Current areas of research interest
- Cell cycle and cell structural responses to change and stress
- Function and regulation of cytoskeletal proteins
- H. pylori (stomach), E. coli (UTI), H. volcanii (archaeal model)
Postgraduate Research - design, analysis, and communication.
Molecular Biology, Microbiology.
Members of the archaeal domain of life that lack homologs of actin and tubulin divide by binary fission in a process that is dependent upon orthologs of eukaryotic ESCRT components. Many of these archaeal organisms are hyperthermophilic acidophiles with unique cell wall structures, which create technical challenges for performing traditional cell biological techniques. Here, we describe the "baby machine" method for synchronizing microorganisms at high temperatures in order to study cell cycle-related processes. We also provide details for fixing, permeabilizing, and staining archaeal cells and ESCRT assemblies for observation by light microscopy.
Walsh, JC, Angstmann, CN, Bisson-Filho, AW, Garner, EC, Duggin, IG & Curmi, PMG 2019, 'Division plane placement in pleomorphic archaea is dynamically coupled to cell shape', Molecular Microbiology.View/Download from: UTS OPUS or Publisher's site
© 2019 John Wiley & Sons Ltd One mechanism for achieving accurate placement of the cell division machinery is via Turing patterns, where nonlinear molecular interactions spontaneously produce spatiotemporal concentration gradients. The resulting patterns are dictated by cell shape. For example, the Min system of Escherichia coli shows spatiotemporal oscillation between cell poles, leaving a mid-cell zone for division. The universality of pattern-forming mechanisms in divisome placement is currently unclear. We examined the location of the division plane in two pleomorphic archaea, Haloferax volcanii and Haloarcula japonica, and showed that it correlates with the predictions of Turing patterning. Time-lapse analysis of H. volcanii shows that divisome locations after successive rounds of division are dynamically determined by daughter cell shape. For H. volcanii, we show that the location of DNA does not influence division plane location, ruling out nucleoid occlusion. Triangular cells provide a stringent test for Turing patterning, where there is a bifurcation in division plane orientation. For the two archaea examined, most triangular cells divide as predicted by a Turing mechanism; however, in some cases multiple division planes are observed resulting in cells dividing into three viable progeny. Our results suggest that the division site placement is consistent with a Turing patterning system in these archaea.
Abdul Halim, MF, Rodriguez, R, Stoltzfus, JD, Duggin, IG & Pohlschroder, M 2018, 'Conserved residues are critical for Haloferax volcanii archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non-homologous sortases from archaea and bacteria.', Molecular microbiology, vol. 108, no. 3, pp. 276-287.View/Download from: UTS OPUS or Publisher's site
Proper protein anchoring is key to the biogenesis of prokaryotic cell surfaces, dynamic, resilient structures that play crucial roles in various cell processes. A novel surface protein anchoring mechanism in Haloferax volcanii depends upon the peptidase archaeosortase A (ArtA) processing C-termini of substrates containing C-terminal tripartite structures and anchoring mature substrates to the cell membrane via intercalation of lipid-modified C-terminal amino acid residues. While this membrane protein lacks clear homology to soluble sortase transpeptidases of Gram-positive bacteria, which also process C-termini of substrates whose C-terminal tripartite structures resemble those of ArtA substrates, archaeosortases do contain conserved cysteine, arginine and arginine/histidine/asparagine residues, reminiscent of His-Cys-Arg residues of sortase catalytic sites. The study presented here shows that ArtAWT -GFP expressed in trans complements ΔartA growth and motility phenotypes, while alanine substitution mutants, Cys173 (C173A), Arg214 (R214A) or Arg253 (R253A), and the serine substitution mutant for Cys173 (C173S), fail to complement these phenotypes. Consistent with sortase active site replacement mutants, ArtAC173A -GFP, ArtAC173S -GFP and ArtAR214A -GFP cannot process substrates, while replacement of the third residue, ArtAR253A -GFP retains some processing activity. These findings support the view that similarities between certain aspects of the structures and functions of the sortases and archaeosortases are the result of convergent evolution.
Mediati, DG, Burke, CM, Ansari, S, Harry, EJ & Duggin, IG 2018, 'High-throughput sequencing of sorted expression libraries reveals inhibitors of bacterial cell division.', BMC genomics, vol. 19, no. 1.View/Download from: UTS OPUS or Publisher's site
BACKGROUND:Bacterial filamentation occurs when rod-shaped bacteria grow without dividing. To identify genetically encoded inhibitors of division that promote filamentation, we used cell sorting flow cytometry to enrich filamentous clones from an inducible expression library, and then identified the cloned DNA with high-throughput DNA sequencing. We applied the method to an expression library made from fragmented genomic DNA of uropathogenic E. coli UTI89, which undergoes extensive reversible filamentation in urinary tract infections and might encode additional regulators of division. RESULTS:We identified 55 genomic regions that reproducibly caused filamentation when expressed from the plasmid vector, and then further localized the cause of filamentation in several of these to specific genes or sub-fragments. Many of the identified genomic fragments encode genes that are known to participate in cell division or its regulation, and others may play previously-unknown roles. Some of the prophage genes identified were previously implicated in cell division arrest. A number of the other fragments encoded potential short transcripts or peptides. CONCLUSIONS:The results provided evidence of potential new links between cell division and distinct cellular processes including central carbon metabolism and gene regulation. Candidate regulators of the UTI-associated filamentation response or others were identified amongst the results. In addition, some genomic fragments that caused filamentation may not have evolved to control cell division, but may have applications as artificial inhibitors. Our approach offers the opportunity to carry out in depth surveys of diverse DNA libraries to identify new genes or sequences encoding the capacity to inhibit division and cause filamentation.
Liao, Y, Ithurbide, S, de Silva, RT, Erdmann, S & Duggin, IG 2018, 'Archaeal cell biology: diverse functions of tubulin-like cytoskeletal proteins at the cell envelope', Emerging Topics in Life Sciences, vol. 2, no. 4, pp. 547-559.View/Download from: UTS OPUS or Publisher's site
Mann, R, Mediati, DG, Duggin, IG, Harry, EJ & Bottomley, AL 2017, 'Metabolic Adaptations of Uropathogenic E. coli in the Urinary Tract.', Frontiers in Cellular and Infection Microbiology, vol. 7, pp. 1-15.View/Download from: UTS OPUS or Publisher's site
Escherichia coli ordinarily resides in the lower gastrointestinal tract in humans, but some strains, known as Uropathogenic E. coli (UPEC), are also adapted to the relatively harsh environment of the urinary tract. Infections of the urine, bladder and kidneys by UPEC may lead to potentially fatal bloodstream infections. To survive this range of conditions, UPEC strains must have broad and flexible metabolic capabilities and efficiently utilize scarce essential nutrients. Whole-organism (or "omics") methods have recently provided significant advances in our understanding of the importance of metabolic adaptation in the success of UPECs. Here we describe the nutritional and metabolic requirements for UPEC infection in these environments, and focus on particular metabolic responses and adaptations of UPEC that appear to be essential for survival in the urinary tract.
Walsh, JC, Angstmann, CN, Duggin, IG & Curmi, PMG 2017, 'Non-linear Min protein interactions generate harmonics that signal mid-cell division in Escherichia coli.', PloS one, vol. 12, no. 10, pp. e0185947-e0185947.View/Download from: UTS OPUS or Publisher's site
The Min protein system creates a dynamic spatial pattern in Escherichia coli cells where the proteins MinD and MinE oscillate from pole to pole. MinD positions MinC, an inhibitor of FtsZ ring formation, contributing to the mid-cell localization of cell division. In this paper, Fourier analysis is used to decompose experimental and model MinD spatial distributions into time-dependent harmonic components. In both experiment and model, the second harmonic component is responsible for producing a mid-cell minimum in MinD concentration. The features of this harmonic are robust in both experiment and model. Fourier analysis reveals a close correspondence between the time-dependent behaviour of the harmonic components in the experimental data and model. Given this, each molecular species in the model was analysed individually. This analysis revealed that membrane-bound MinD dimer shows the mid-cell minimum with the highest contrast when averaged over time, carrying the strongest signal for positioning the cell division ring. This concurs with previous data showing that the MinD dimer binds to MinC inhibiting FtsZ ring formation. These results show that non-linear interactions of Min proteins are essential for producing the mid-cell positioning signal via the generation of second-order harmonic components in the time-dependent spatial protein distribution.
Boysen, A, Palmisano, G, Krogh, TJ, Duggin, IG, Larsen, MR & Moller-Jensen, J 2016, 'A novel mass spectrometric strategy "BEMAP" reveals Extensive O-linked protein glycosylation in Enterotoxigenic Escherichia coli', SCIENTIFIC REPORTS, vol. 6.View/Download from: UTS OPUS or Publisher's site
Liao, Y, Williams, TJ, Walsh, JC, Ji, M, Poljak, A, Curmi, PMG, Duggin, IG & Cavicchioli, R 2016, 'Developing a genetic manipulation system for the Antarctic archaeon, Halorubrum lacusprofundi: investigating acetamidase gene function.', Scientific Reports, vol. 6, pp. 1-15.View/Download from: UTS OPUS or Publisher's site
No systems have been reported for genetic manipulation of cold-adapted Archaea. Halorubrum lacusprofundi is an important member of Deep Lake, Antarctica (~10% of the population), and is amendable to laboratory cultivation. Here we report the development of a shuttle-vector and targeted gene-knockout system for this species. To investigate the function of acetamidase/formamidase genes, a class of genes not experimentally studied in Archaea, the acetamidase gene, amd3, was disrupted. The wild-type grew on acetamide as a sole source of carbon and nitrogen, but the mutant did not. Acetamidase/formamidase genes were found to form three distinct clades within a broad distribution of Archaea and Bacteria. Genes were present within lineages characterized by aerobic growth in low nutrient environments (e.g. haloarchaea, Starkeya) but absent from lineages containing anaerobes or facultative anaerobes (e.g. methanogens, Epsilonproteobacteria) or parasites of animals and plants (e.g. Chlamydiae). While acetamide is not a well characterized natural substrate, the build-up of plastic pollutants in the environment provides a potential source of introduced acetamide. In view of the extent and pattern of distribution of acetamidase/formamidase sequences within Archaea and Bacteria, we speculate that acetamide from plastics may promote the selection of amd/fmd genes in an increasing number of environmental microorganisms.
Walsh, JC, Angstmann, CN, McGann, AV, Henry, BI, Duggin, IG & Curmi, PMG 2016, 'Patterning of the MinD cell division protein in cells of arbitrary shape can be predicted using a heuristic dispersion relation', AIMS Biophysics, vol. 3, no. 1, pp. 119-145.View/Download from: UTS OPUS or Publisher's site
© 2016, Paul M. G. Curmi, et al. Many important cellular processes require the accurate positioning of subcellular structures. Underpinning many of these are protein systems that spontaneously generate spatiotemporal patterns. In some cases, these systems can be described by non-linear reaction-diffusion equations, however, a full description of such equations is rarely available. A well-studied patterning system is the Min protein system that underpins the positioning of the FtsZ contractile ring during cell division in Escherichia coli. Using a coordinate-free linear stability analysis, the reaction terms can be separated from the geometry of a cell. The reaction terms produce a dispersion relation that can be used to predict patterning on any cell shape and size. Applying linear stability analysis to an accurate mathematical model of the Min system shows that while it correctly predicts the onset of patterning, the dispersion relation fails to predict oscillations and quantitative mode transitions. However, we show that data from full solutions of the Min model can be used to generate a heuristic dispersion relation. We show that this heuristic dispersion relation can be used to approximate the Min protein patterning obtained by full simulations of the non-linear reaction-diffusion equations. Moreover, it also predicts Min patterning obtained from experiments where the shapes of E. coli cells have been deformed into rectangles or arbitrary shapes. Using this procedure, it should be possible to generate heuristic dispersion relations from protein patterning data or simulations for any patterning process and subsequently use these to predict patterning for arbitrary cell shapes.
Duggin, IG, Aylett, CHS, Walsh, JC, Michie, KA, Wang, Q, Turnbull, L, Dawson, EM, Harry, EJ, Whitchurch, CB, Amos, LA & Loewe, J 2015, 'CetZ tubulin-like proteins control archaeal cell shape', NATURE, vol. 519, no. 7543, pp. 362-+.View/Download from: UTS OPUS or Publisher's site
Walsh, JC, Angstmann, CN, Duggin, IG & Curmi, PMG 2015, 'Molecular Interactions of the Min Protein System Reproduce Spatiotemporal Patterning in Growing and Dividing Escherichia coli Cells', PLOS ONE, vol. 10, no. 5.View/Download from: UTS OPUS or Publisher's site
Delmas, S, Duggin, IG & Allers, T 2013, 'DNA damage induces nucleoid compaction via the Mre11-Rad50 complex in the archaeon Haloferax volcanii.', Molecular Microbiology, vol. 87, no. 1, pp. 168-179.View/Download from: UTS OPUS or Publisher's site
In prokaryotes the genome is organized in a dynamic structure called the nucleoid, which is embedded in the cytoplasm. We show here that in the archaeon Haloferax volcanii, compaction and reorganization of the nucleoid is induced by stresses that damage the genome or interfere with its replication. The fraction of cells exhibiting nucleoid compaction was proportional to the dose of the DNA damaging agent, and results obtained in cells defective for nucleotide excision repair suggest that breakage of DNA strands triggers reorganization of the nucleoid. We observed that compaction depends on the Mre11-Rad50 complex, suggesting a link to DNA double-strand break repair. However, compaction was observed in a radA mutant, indicating that the role of Mre11-Rad50 in nucleoid reorganisation is independent of homologous recombination. We therefore propose that nucleoid compaction is part of a DNA damage response that accelerates cell recovery by helping DNA repair proteins to locate their targets, and facilitating the search for intact DNA sequences during homologous recombination
Brymora, A, Duggin, IG, Berven, LA, van Dam, EM, Roufogalis, BD & Robinson, PJ 2012, 'Identification and characterisation of the RalA-ERp57 interaction: evidence for GDI activity of ERp57.', PLoS ONE, vol. 7, no. 11, pp. 1-11.View/Download from: UTS OPUS or Publisher's site
RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro. These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57's active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H(2)O(2) inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor (RalGDI).
Gristwood, T, Duggin, IG, Wagner, M, Albers, SV & Bell, SD 2012, 'The sub-cellular localization of Sulfolobus DNA replication', Nucleic Acids Research, vol. 40, no. 12, pp. 5487-5496.View/Download from: UTS OPUS or Publisher's site
Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.
Duggin, IG, Dubarry, N & Bell, SD 2011, 'Replication termination and chromosome dimer resolution in the archaeon Sulfolobus solfataricus', The EMBO Journal, vol. 30, no. 1, pp. 145-153.View/Download from: UTS OPUS or Publisher's site
Archaea of the genus Sulfolobus have a single-circular chromosome with three replication origins. All three origins fire in every cell in every cell cycle. Thus, three pairs of replication forks converge and terminate in each replication cycle. Here, we report 2D gel analyses of the replication fork fusion zones located between origins. These indicate that replication termination involves stochastic fork collision. In bacteria, replication termination is linked to chromosome dimer resolution, a process that requires the XerC and D recombinases, FtsK and the chromosomal dif site.
Duggin, IG & Bell, SD 2009, 'Termination structures in the Escherichia coli chromosome replication fork trap', Journal of Molecular Biology, vol. 387, no. 3, pp. 532-539.View/Download from: UTS OPUS or Publisher's site
The Escherichia coli chromosome contains two opposed sets of unidirectional DNA replication pause (Ter) sites that, according to the replication fork trap theory, control the termination of chromosome replication by restricting replication fork fusion to the terminus region. In contrast, a recent hypothesis suggested that termination occurs at the dif locus instead. Using twodimensional agarose gel electrophoresis, we examined DNA replication intermediates at the Ter sites and at dif in wild-type cells. Two definitive signatures of site-specific terminationspecific replication fork arrest and converging replication forkswere clearly detected at Ter sites, but not at dif. We also detected a significant pause during the latter stages of replication fork convergence at Ter sites. Quantification of fork pausing at the Ter sites in both their native chromosomal context and the plasmid context further supported the fork trap model.
Duggin, IG, McCallum, SA & Bell, SD 2008, 'Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius', Proceedings of the National Academy of Sciences, vol. 105, no. 43, pp. 16737-16742.View/Download from: UTS OPUS or Publisher's site
The ``baby machine provides a means of generating synchronized cultures of minimally perturbed cells. We describe the use of this technique to establish the key cell-cycle parameters of hyperthermophilic archaea of the genus Sulfolobus. The 3 DNA replication origins of Sulfolobus acidocaldarius were mapped by 2D gel analysis to near 0 (oriC2), 579 (oriC1), and 1,197 kb (oriC3) on the 2,226-kb circular genome, and we present a direct demonstration of their activity within the first few minutes of a synchronous cell cycle. We also detected X-shaped DNA molecules at the origins in log-phase cells, but these were not directly associated with replication initiation or ongoing chromosome replication in synchronized cells. Whole-genome marker frequency analyses of both synchronous and log-phase cultures showed that origin utilization was close to 100% for all 3 origins per round of replication. However, oriC2 was activated slightly later on average compared with oriC1 and oriC3. The DNA replication forks moved bidirectionally away from each origin at 88 bp per second in synchronous culture. Analysis of the 3 Orc1/Cdc6 initiator proteins showed a uniformity of cellular abundance and origin binding throughout the cell cycle. In contrast, although levels of theMCMhelicase were constant across the cell cycle, its origin localization was regulated, because it was strongly enriched at all 3 origins in early S phase.
Duggin, IG, Wake, RG, Bell, SD & Hill, TM 2008, 'The replication fork trap and termination of chromosome replication', Molecular Microbiology, vol. 70, no. 6, pp. 1323-1333.View/Download from: UTS OPUS or Publisher's site
Bacteria that have a circular chromosome with a bidirectional DNA replication origin are thought to utilize a `replication fork trap to control termination of replication. The fork trap is an arrangement of replication pause sites that ensures that the two replication forks fuse within the terminus region of the chromosome, approximately opposite the origin on the circular map. However, the biological significance of the replication fork trap has been mysterious, as its inactivation has no obvious consequence. Here we review the research that led to the replication fork trap theory, and we aim to integrate several recent findings that contribute towards an understanding of the physiological roles of the replication fork trap. Likely roles include the prevention of over-replication, and the optimization of post-replicative mechanisms of chromosome segregation, such as that involving FtsK in Escherichia coli.
Frols, S, Gordon, PM, Panlilio, MA, Duggin, IG, Bell, SD, Sensen, CW & Schleper, C 2007, 'Response of the hyperthermophilic archaeon Sulfolobus solfataricus to UV damage', Journal Of Bacteriology, vol. 189, no. 23, pp. 8708-8718.View/Download from: UTS OPUS or Publisher's site
In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.
Fu, D, van Dam, EM, Duggin, IG, Brymora, A & Robinson, PJ 2007, 'The small GTPases Rab5 and RalA regulate intracellular traffic of P-glycoprotein', Biochimica et Biophysica Acta, vol. 1773, pp. 1062-1072.View/Download from: UTS OPUS
P-glycoprotein (P-gp) is a plasma membrane glycoprotein that can cause multidrug resistance (MDR) of cancer cells by acting as an ATP-dependent drug efflux pump. The regulatory effects of the small GTPases Rab5 and RalA on the intracellular trafficking of P-gp were investigated in HeLa cells. As expected, overexpressed enhanced green fluorescent protein (EGFP)-tagged P-gp (P-gp-EGFP) is mainly localised to the plasma membrane. However, upon cotransfection of either dominant negative Rab5 (Rab5-S34N) or constitutively active RalA (RalA-G23V) the intracellular P-gp-EGFP levels increased approximately 9 and 13 fold, respectively, compared to control P-gp-EGFP cells. These results suggest that Rab5 and RalA regulate P-gp trafficking between the plasma membrane and an intracellular compartment. In contrast, coexpression of constitutively active Rab5 (Rab5-Q79L) or dominant negative RalA (RalA-S28N) had no effect on the localisation of P-gp-EGFP. Furthermore, the intracellular accumulation of daunorubicin, a substrate for P-gp, increased significantly with an increased intracellular localisation of P-gp-EGFP. These results imply that it may be possible to overcome MDR by controlling the plasma membrane localisation of P-gp.
Duggin, IG 2006, 'DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding', Journal of Molecular Biology, vol. 361, no. 1, pp. 1-6.View/Download from: UTS OPUS or Publisher's site
In order to elucidate the mechanism of DNA replication fork arrest by the replication terminator protein (RTP)DNA complex, a set of RTP fusion proteins were constructed in which peptides of various sizes were fused to the C terminus; this placed the peptides at a surface location that was predicted to come into contact with the DNA replication machinery during fork arrest. The fusion proteins were capable of replication fork arrest in vivo, but they had a significantly reduced efficiency compared to wild-type RTP, which was not directly proportional to peptide size or sequence. Importantly, the fusion proteins retained completely normal RTPDNA binding affinity. These findings rule out the molecular clamp model as the sole explanation for fork arrest by RTP, and suggest that RTP interacts with the replication machinery in a manner that directly contributes to the fork arrest mechanism.
Duggin, IG & Bell, SD 2006, 'The Chromosome Replication Machinery of the Archaeon Sulfolobus solfataricus', Journal Of Biological Chemistry, vol. 281, no. 22, pp. 15029-15032.View/Download from: Publisher's site
In the three domains of life, the archaea, bacteria, and eukarya, there are two general lineages of DNA replication proteins: the bacterial and the eukaryal/archaeal lineages. The hyperthermophilic archaeon Sulfolobus solfataricus provides an attractive model for biochemical study of DNA replication. Its relative simplicity in both genomic and biochemical contexts, together with high protein thermostability, has already provided insight into the function of the more complex yet homologous molecules of the eukaryotic domain. Here, we provide an overview of recent insights into the functioning of the chromosome replication machinery of S. solfataricus, focusing on some of the relatively well characterized core components that act at the DNA replication fork.
Duggin, IG, Matthews, JM, Dixon, NE, Wake, RG & Mackay, JP 2005, 'A Complex Mechanism Determines Polarity of DNA Replication Fork Arrest by the Replication Terminator Complex of Bacillus subtilis', Journal Of Biological Chemistry, vol. 280, no. 13, pp. 13105-13113.View/Download from: UTS OPUS or Publisher's site
Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this differential binding affinity model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA
Hastings, AF, Otting, G, Folmer, RH, Duggin, IG, Wake, RG, Wilce, MC & Wilce, JA 2005, 'Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy', Biochemical and Biophysical Research Communication, vol. 335, pp. 361-366.View/Download from: UTS OPUS or Publisher's site
Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H15N spectra revealed that the N-termini, ?3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the proteinDNA interface
Duggin, IG 2004, 'Studying DNA terminator proteins: a means to an end', Australian Biochemist, vol. 35, no. 1, pp. 5-8.
Vivian, JP, Hastings, AF, Duggin, IG, Wake, RG, Wilce, MJ & Wilce, JA 2003, 'The impact of single cysteine residue mutations on the replication terminator protein', Biochemical and Biophysical Research Communication, vol. 310, no. 4, pp. 1096-1103.View/Download from: UTS OPUS or Publisher's site
We report the structural and biophysical consequences of cysteine substitutions in the DNA-binding replication terminator protein (RTP) of Bacillus subtilis, that resulted in an optimised RTP mutant suitable for structural studies. The cysteine residue 110 was replaced with alanine, valine or serine. Protein secondary structure and stability (using circular dichroism spectropolarimetry), self-association (using analytical ultracentrifugation), and DNA-binding measurements revealed RTP.C110S to be the most similar mutant to wild-type RTP. The C110A and C110V.RTP mutants were less soluble, less stable and showed lower DNA-binding affinity. The structure of RTP.C110S, solved to 2.5AA resolution using crystallographic methods, showed no major structural perturbation due to the mutation. Heteronuclear NMR spectroscopic studies revealed subtle differences in the electronic environment about the site of mutation. The study demonstrates the suitability of serine as a substitute for cysteine in RTP and the high sensitivity of protein behaviour to single amino acid substitutions.
Wilce, JA, Vivian, JP, Hastings, AF, Otting, G, Folmer, RH, Duggin, IG, Wake, RG & Wilce, MJ 2001, 'Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest', Nature Structural Biology, vol. 8, no. 3, pp. 206-210.View/Download from: UTS OPUS or Publisher's site
The coordinated termination of DNA replication is an important step in the life cycle of bacteria with circular chromosomes, but has only been defined at a molecular level in two systems to date. Here we report the structure of an engineered replication terminator protein (RTP) of Bacillus subtilis in complex with a 21 base pair DNA by X-ray crystallography at 2.5 Å resolution.
Andersen, PA, Griffiths, AA, Duggin, IG & Wake, RG 2000, 'Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for TusÂ±Ter functioning in E. coli', Molecular Microbiology, vol. 36, no. 6, pp. 1327-1335.View/Download from: UTS OPUS or Publisher's site
The Escherichia coli replication terminator TerB was inserted in its two alternate orientations into a Bacillus subtilis fork-arrest assay plasmid. After transferring these new plasmids into B. subtilis, which could overproduce the E. coli terminator protein Tus, it was shown that the E. coli Tus±TerB complex could cause polar replication fork arrest, albeit at a very low level, in B. subtilis. A new B. subtilis±E. coli shuttle plasmid was designed to allow the insertion of either the TerI (B. subtilis) or TerB (E. coli) terminator at the same site and in the active orientation in relation to the approaching replication fork generated in either organism. Fork-arrest assays for both terminator-containing plasmids replicating in both organisms which also produced saturating levels of either the B. subtilis terminator protein (RTP) or Tus were performed
Duggin, IG, Andersen, PA, Smith, MT, Wilce, JA, King, GF & Wake, RG 1999, 'Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest', Journal of Molecular Biology, vol. 286, no. 5, pp. 1325-1335.View/Download from: UTS OPUS or Publisher's site
DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to speci®c DNA terminator sequences (Ter sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a speci®c interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. In support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona ®de in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation.
In comparison with bacteria and eukaryotes, the large and diverse group of microorganisms known as archaea possess a great diversity of cytoskeletal proteins, including members of the tubulin superfamily. Many species contain FtsZ, CetZ and even possible tubulins; however, some major taxonomic groups do not contain any member of the tubulin superfamily. Studies using the model archaeon, Halferax volcanii have recently been instrumental in defining the fundamental roles of FtsZ and CetZ in archaeal cell division and cell shape regulation. Structural studies of archaeal tubulin superfamily proteins provide a definitive contribution to the cytoskeletal field, showing which protein-types must have developed prior to the divergence of archaea and eukaryotes. Several regions of the globular core domain - the "signature" motifs - combine in the 3D structure of the common molecular fold to form the GTP-binding site. They are the most conserved sequence elements and provide the primary basis for identification of new superfamily members through homology searches. The currently well-characterised proteins also all share a common mechanism of GTP-dependent polymerisation, in which GTP molecules are sandwiched between successive subunits that are arranged in a head-to-tail manner. However, some poorly-characterised archaeal protein families retain only some of the signature motifs and are unlikely to be capable of dynamic polymerisation, since the promotion of depolymerisation by hydrolysis to GDP depends on contributions from both subunits that sandwich the nucleotide in the polymer.
n the case of a circular bacterial chromosome, termination of DNA replication occurs when the two replication forks, progressing in opposite directions, meet and fuse in a specific region of the chromosome, which is generally diametrically opposed to the site of initiation of DNA replication. Most research has focused on the systems utilized by the rod-shaped Gram-negative Escherichia coli and Gram-positive Bacillus subtilis.
Duggin, IG & Wake, RG 2002, 'Termination of Chromosome Replication' in Sonenshein, AL (ed), Bacillus subtilis and its closest relatives: from genes to cells, ASM Press, USA, pp. 87-95.