ORCID Profile
0000-0002-6628-876X
Current Organisation
University of Wollongong
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Enzymes | Biochemistry and Cell Biology | Structural Biology (incl. Macromolecular Modelling) | Bacteriology
Expanding Knowledge in the Biological Sciences | Human Pharmaceutical Treatments (e.g. Antibiotics) |
Publisher: Elsevier BV
Date: 03-2011
Publisher: Cold Spring Harbor Laboratory
Date: 10-09-2018
DOI: 10.1101/413880
Abstract: The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.
Publisher: Cold Spring Harbor Laboratory
Date: 08-01-2019
DOI: 10.1101/515536
Abstract: Actively transcribed genes are preferentially repaired in a conserved repair reaction known as transcription-coupled nucleotide excision repair 1–3 . During this reaction, stalled transcription elongation complexes at sites of lesions serve as a signal to trigger the assembly of nucleotide excision repair factors (reviewed in ref. 4,5 ). In the model organism Escherichia coli , the transcription-repair coupling factor Mfd displaces the stalled RNA polymerase and hands-off the stall site to the nucleotide excision repair factors UvrAB for damage detection 6–9 . Despite in vitro evidence, it remains unclear how in live cells the stall site is faithfully handed over to UvrB from RNA polymerase and whether this handoff occurs via the Mfd-UvrA 2 -UvrB complex or via alternate reaction intermediates. Here, we visualise Mfd, the central player of transcription-coupled repair in actively growing cells and determine the catalytic requirements for faithful completion of the handoff during transcription-coupled repair. We find that the Mfd-UvrA 2 complex is arrested on DNA in the absence of UvrB. Further, Mfd-UvrA 2 -UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition, were also impaired in successful handoff. Our observations demonstrate that in live cells, the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner during transcription-coupled repair.
Publisher: eLife Sciences Publications, Ltd
Date: 05-02-2019
DOI: 10.7554/ELIFE.42761
Abstract: The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.
Publisher: Oxford University Press (OUP)
Date: 19-03-2013
DOI: 10.1093/NAR/GKT177
Publisher: Portland Press Ltd.
Date: 12-2017
DOI: 10.1042/BST20170055
Abstract: Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow in idual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA–repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA–repair processes in prokaryotes.
Publisher: Cold Spring Harbor Laboratory
Date: 29-04-2019
DOI: 10.1101/620195
Abstract: DNA polymerase IV (pol IV) is expressed at increased levels in Escherichia coli cells suffering high levels of DNA damage. In a recent single-molecule imaging study, we demonstrated that elevating the pol IV concentration is not sufficient to provide access to binding sites on the nucleoid, suggesting that other factors may recruit pol IV to its substrates once the DNA becomes damaged. Here we extend this work, investigating the proteins UmuD and RecA as potential modulators of pol IV activity. UmuD promotes long-lived association of pol IV with the nucleoid, whereas its cleaved form, UmuD’, which accumulates in DNA-damaged cells, inhibits binding. In agreement with proposed roles for pol IV in homologous recombination, up to 40% of pol IV foci colocalise with a probe for RecA* nucleoprotein filaments in ciprofloxacin-treated cells. A hyperactive RecA mutant, recA (E38K), allows pol IV to bind the nucleoid even in the absence of exogenous DNA damage. In vitro, RecA(E38K) forms RecA*-like structures that can recruit pol IV, even on double-stranded DNA, consistent with a physical interaction between RecA and pol IV. Together, the results indicate that UmuD and RecA modulate the binding of pol IV to its DNA substrates, which frequently coincide with RecA* structures.
Publisher: American Chemical Society (ACS)
Date: 29-04-2013
DOI: 10.1021/NN305787M
Abstract: Nanotechnology based on the highly specific pairing of nucleobases in DNA has been used to generate a wide variety of well-defined two- and three-dimensional assemblies, both static and dynamic. However, control over the junction angles to achieve them has been limited. To achieve higher order assemblies, the strands of the DNA duplex are typically made to deviate at junctions with configurations based on crossovers or non-DNA moieties. Such strand crossovers tend to be intrinsically unstructured with the overall structural rigidity determined by the architecture of the nanoassembly, rather than the junction itself. Specific approaches to define nanoassembly junction angles are based either on the cooperative twist- and strain-promoted tuning of DNA persistence length leading to bent DNA rods for fairly large nano-objects, or de novo synthesis of in idual junction inserts that are typically non-DNA and based on small organic molecules or metal-coordinating ligand moieties. Here, we describe a general strategy for direct control of junction angles in DNA nanostructures that are completely tunable about the DNA helix. This approach is used to define angular vertices through readily accessible backbone-branched DNAs (bbDNAs). We demonstrate how such bbDNAs can be used as a new building block in DNA nanoconstruction to obtain well-defined nanostructures. Angular control through readily accessible bbDNA building block provides a general and versatile approach for incorporating well-defined junctions in nanoconstructs and expands the toolkit toward achieving strain free, highly size- and shape-tunable DNA based architectures.
Publisher: Oxford University Press (OUP)
Date: 04-03-2013
DOI: 10.1093/NAR/GKT138
Publisher: Springer Science and Business Media LLC
Date: 20-03-2020
DOI: 10.1038/S41467-020-15182-3
Abstract: The Escherichia coli transcription-repair coupling factor Mfd displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Whether this handoff from RNA polymerase to UvrA occurs via the Mfd-UvrA 2 -UvrB complex or alternate reaction intermediates in cells remains unclear. Here, we visualise Mfd in actively growing cells and determine the catalytic requirements for faithful recruitment of nucleotide excision repair proteins. We find that ATP hydrolysis by UvrA governs formation and disassembly of the Mfd-UvrA 2 complex. Further, Mfd-UvrA 2 -UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition are impaired in successful handoff. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells shows that the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner.
Publisher: Cold Spring Harbor Laboratory
Date: 21-07-2023
DOI: 10.1101/2023.07.20.549978
Abstract: Ring-shaped DNA sliding cl s are essential for DNA replication and genome maintenance. Cl s need to be opened or trapped open and chaperoned onto DNA by cl loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place cl s around DNA remains limited. Here, we present a series of six structures of the Escherichia coli CLC bound to an open or closed cl on and off a primer-template DNA that represent all intermediates in the cl loading process. We show that the ATP-bound CLC first binds to a cl , then constricts to hold onto it. The CLC then expands to open the cl with a gap large enough for double-stranded DNA to enter. Upon binding to DNA, the CLC constricts slightly, allowing ATP hydrolysis and cl closing around DNA. Although both yeast and E. coli CLCs open cl s by crab claw-like motions, they do it by the CLC expanding in opposite directions. These structures provide critical high-resolution snapshots of cl loading by the E. coli CLC, revealing how the molecular machine works.
Publisher: Oxford University Press (OUP)
Date: 20-03-2023
DOI: 10.1093/NAR/GKAD186
Abstract: Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by in idual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging in idual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.
Publisher: Springer Science and Business Media LLC
Date: 05-08-2014
Publisher: Springer Science and Business Media LLC
Date: 20-04-2018
DOI: 10.1038/S41467-018-03790-Z
Abstract: During transcription elongation, bacterial RNA polymerase (RNAP) can pause, backtrack or stall when transcribing template DNA. Stalled transcription elongation complexes at sites of bulky lesions can be rescued by the transcription terminator Mfd. The molecular mechanisms of Mfd recruitment to transcription complexes in vivo remain to be elucidated, however. Using single-molecule live-cell imaging, we show that Mfd associates with elongation transcription complexes even in the absence of exogenous genotoxic stresses. This interaction requires an intact RNA polymerase-interacting domain of Mfd. In the presence of drugs that stall RNAP, we find that Mfd associates pervasively with RNAP. The residence time of Mfd foci reduces from 30 to 18 s in the presence of endogenous UvrA, suggesting that UvrA promotes the resolution of Mfd-RNAP complexes on DNA. Our results reveal that RNAP is frequently rescued by Mfd during normal growth and highlight a ubiquitous house-keeping role for Mfd in regulating transcription elongation.
Publisher: Elsevier BV
Date: 12-2019
Publisher: Elsevier BV
Date: 12-2021
Publisher: Springer Science and Business Media LLC
Date: 20-03-2020
DOI: 10.1038/S41467-020-15179-Y
Abstract: In the model organism Escherichia coli , helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled by lesions. Ultimately, damage recognition is mediated by UvrA, followed by verification by UvrB. Here we characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB by following functional, fluorescently tagged UvrA molecules in live TCR-deficient or wild-type cells. The lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, the lifetimes of UvrA strongly depended on, and matched those of Mfd. Overall, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.
Publisher: Oxford University Press (OUP)
Date: 20-07-2020
DOI: 10.1093/NAR/GKAA597
Abstract: Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.
Publisher: Oxford University Press (OUP)
Date: 18-01-2019
DOI: 10.1093/NAR/GKZ003
Publisher: Elsevier BV
Date: 10-2010
Publisher: Springer Science and Business Media LLC
Date: 16-09-2019
DOI: 10.1038/S41598-019-49837-Z
Abstract: Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro . We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro .
Publisher: Cold Spring Harbor Laboratory
Date: 08-01-2019
DOI: 10.1101/515502
Abstract: In the model organism Escherichia coli , helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled or paused by lesions. Ultimately, damage recognition is mediated by UvrA, culminating in the loading of the damage verification enzyme UvrB. We set out to characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB. We followed functional, fluorescently tagged UvrA molecules in live cells and measured their residence times in TCR-deficient or wild-type cells. We demonstrate that the lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, we found that the lifetimes of UvrA strongly depended on, and matched those of Mfd. Here, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.
Publisher: Elsevier BV
Date: 03-2018
DOI: 10.1016/J.TIBS.2017.12.004
Abstract: Cells use a suite of specialized enzymes to repair chromosomal double-strand breaks (DSBs). Two recent studies describe how single-molecule fluorescence imaging techniques are used in the direct visualization of some of the key molecular steps involved. De Tullio et al. and Kaniecki et al. watch in idual Srs2 helicase molecules disrupt repair intermediates formed by RPA, Rad51, and Rad52 on DNA during homologous recombination.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5MB00321K
Abstract: iSBatch: an ImageJ plugin for fast evaluation of analysis pipelines and visual exploration of datasets.
Publisher: Wiley
Date: 23-03-2007
Publisher: Proceedings of the National Academy of Sciences
Date: 23-04-2014
Abstract: UV damage in genomic DNA is identified by the human UV-damaged DNA-binding protein (UV-DDB). Recognition of DNA damage by UV-DDB serves to initiate global genomic nucleotide excision repair (NER) in humans. Recent work has revealed that UV-DDB dimerizes at sites of damage. This study demonstrates that prior to stable damage recognition, UV-DDB interrogates DNA for damage via a 3D diffusion mechanism coupled to the formation of multiple transient intermediates. Stable binding at sites of damage is achieved by dimerization of UV-DDB. This study also analyzed a disease-causing mutant of UV-DDB, which was found to slide on DNA, while retaining the ability to dimerize on DNA. These results enhance our understanding of damage recognition in NER in humans.
Publisher: Proceedings of the National Academy of Sciences
Date: 20-07-2012
Abstract: UV light-induced photoproducts are recognized and removed by the nucleotide-excision repair (NER) pathway. In humans, the UV-damaged DNA-binding protein (UV-DDB) is part of a ubiquitin E3 ligase complex (DDB1-CUL4A DDB2 ) that initiates NER by recognizing damaged chromatin with concomitant ubiquitination of core histones at the lesion. We report the X-ray crystal structure of the human UV-DDB in a complex with damaged DNA and show that the N-terminal domain of DDB2 makes critical contacts with two molecules of DNA, driving N-terminal-domain folding and promoting UV-DDB dimerization. The functional significance of the dimeric UV-DDB [(DDB1-DDB2) 2 ], in a complex with damaged DNA, is validated by electron microscopy, atomic force microscopy, solution biophysical, and functional analyses. We propose that the binding of UV-damaged DNA results in conformational changes in the N-terminal domain of DDB2, inducing helical folding in the context of the bound DNA and inducing dimerization as a function of nucleotide binding. The temporal and spatial interplay between domain ordering and dimerization provides an elegant molecular rationale for the unprecedented binding affinities and selectivities exhibited by UV-DDB for UV-damaged DNA. Modeling the DDB1-CUL4A DDB2 complex according to the dimeric UV-DDB-AP24 architecture results in a mechanistically consistent alignment of the E3 ligase bound to a nucleosome harboring damaged DNA. Our findings provide unique structural and conformational insights into the molecular architecture of the DDB1-CUL4A DDB2 E3 ligase, with significant implications for the regulation and overall organization of the proteins responsible for initiation of NER in the context of chromatin and for the consequent maintenance of genomic integrity.
Publisher: eLife Sciences Publications, Ltd
Date: 08-01-2019
Publisher: Elsevier BV
Date: 09-2019
Publisher: Elsevier BV
Date: 07-2016
Publisher: Oxford University Press (OUP)
Date: 31-05-2022
DOI: 10.1093/NAR/GKAC449
Abstract: Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.
Publisher: Cold Spring Harbor Laboratory
Date: 05-12-2022
DOI: 10.1101/2022.12.04.519054
Abstract: Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli , these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by in idual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging in idual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that replication restart is the rate-limiting step of stalled replication rescue.
Publisher: Public Library of Science (PLoS)
Date: 28-08-2015
Location: Netherlands
Start Date: 02-2021
End Date: 02-2024
Amount: $692,195.00
Funder: Australian Research Council
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