ORCID Profile
0000-0002-9945-6133
Current Organisation
The Francis Crick Institute
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Characterisation of biological macromolecules | Enzymes | Biochemistry and cell biology | Structural biology (incl. macromolecular modelling) |
Publisher: Elsevier BV
Date: 03-2020
Abstract: Many strategies have been developed to manipulate DNA molecules and investigate protein-DNA interactions with single-molecule resolution. Often, these require long DNA molecules with a length of several 10s of kb that are chemically modified at specific regions. This need has traditionally been met by commercially available DNA from bacteriophage λ. However, λ DNA does not allow for the generation of highly customizable substrates in a straightforward manner, an important factor when developing assays to study complex biochemical reactions. Here we present a generalizable method for the design and production of very long chemically modified DNA substrates derived from a single plasmid. We show the versatility of this design by demonstrating its application in studying DNA replication in vitro. We anticipate this strategy will be broadly useful in producing a range of long chemically modified DNA molecules required for a erse range of single-molecule approaches.
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: MDPI AG
Date: 30-01-2019
DOI: 10.3390/MOLECULES24030491
Abstract: The ability to watch single molecules of DNA has revolutionised how we study biological transactions concerning nucleic acids. Many strategies have been developed to manipulate DNA molecules to investigate mechanical properties, dynamics and protein–DNA interactions. Imaging methods using small molecules and protein-based probes to visualise DNA have propelled our understanding of complex biochemical reactions involving DNA. This review focuses on summarising some of the methodological developments made to visualise in idual DNA molecules and discusses how these probes have been used in single-molecule biophysical assays.
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: Portland Press Ltd.
Date: 05-05-2020
DOI: 10.1042/BST20190998
Abstract: This review discusses recent advances in single-particle cryo-EM and single-molecule approaches used to visualise eukaryotic DNA replication reactions reconstituted in vitro. We comment on the new challenges facing structural biologists, as they turn to describing the dynamic cascade of events that lead to replication origin activation and fork progression.
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: Proceedings of the National Academy of Sciences
Date: 18-09-2017
Abstract: Replication of genomic DNA is essential to all cells. The replisome, the multiprotein machine that performs DNA replication, contains many moving parts, the actions of which are poorly understood. Unraveling the dynamic behavior of these proteins requires novel application of single-molecule imaging techniques to eliminate averaging inherent in ensemble methods and to directly observe short-lived events. Here, we present single-molecule observations of an active Saccharomyces cerevisiae replisome using purified proteins. We find that a checkpoint complex (Mrc1–Tof1–Csm3), known to bind and to speed up the replisome, interacts only transiently with the replisome. This work represents a major step toward establishing the tools needed to understand the detailed kinetics of proteins within the complex eukaryotic replisome.
Publisher: Cold Spring Harbor Laboratory
Date: 08-04-2019
DOI: 10.1101/602086
Abstract: Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α DNA polymerase activity in replication and show that Pol α primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.
Publisher: Proceedings of the National Academy of Sciences
Date: 16-11-2020
Abstract: The replisome machine that duplicates the DNA genome encounters a variety of blocks to replication, such as DNA bound proteins, R-loops, and DNA lesions. Studies in bacterial systems demonstrate that replisome advance through blocks is facilitated by an accessory monomeric helicase that acts on the opposite strand from the replicative hexameric helicase. This report examines this issue in a eukaryotic system, using Pif1, a monomeric helicase that acts on the opposite strand from the replicative CMG helicase. The report shows that an inactive “dead” Cas9 (dCas9) protein R-loop block arrests the replisome, but Pif1 enables replisome bypass of the dCas9 R-loop block. Hence, use of monomeric helicases may have evolved to aid replisome bypass of protein-DNA and protein-bound R-loop blocks.
Publisher: Cold Spring Harbor Laboratory
Date: 07-07-2021
DOI: 10.1101/2021.07.07.451541
Abstract: The replisome is responsible for replication of DNA in all domains of life, with several of its in idual enzyme components relying on hydrolysis of nucleoside triphosphates to provide energy for replisome function. Half a century of biochemical studies have demonstrated a dependence on ATP as an energy source for helicases to unwind duplex DNA during replication. Through single-molecule visualization of DNA replication by the Escherichia coli replisome, we demonstrate that the DnaB helicase does not rely on hydrolysis of ATP (or any ribo-NTPs) in the context of the elongating replisome. We establish that nucleotide incorporation by the leading-strand polymerase is the main motor driving the replication process. Polymerases provide the energy for helicase-mediated DNA unwinding during E. coli DNA replication.
Publisher: eLife Sciences Publications, Ltd
Date: 22-04-2017
DOI: 10.7554/ELIFE.23932
Abstract: The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding cl ), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled cl loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.
Publisher: Elsevier BV
Date: 07-2020
Publisher: eLife Sciences Publications, Ltd
Date: 08-01-2019
Publisher: Springer Science and Business Media LLC
Date: 15-06-2022
DOI: 10.1038/S41586-022-04829-4
Abstract: The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45–MCM–GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown 1 . Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.
Publisher: Elsevier
Date: 2022
DOI: 10.1016/BS.MIE.2022.02.022
Abstract: Single-molecule imaging studies using long linear DNA substrates have revealed unanticipated insights into the dynamics of multi-protein systems. The use of long DNA substrates allows for the study of protein-DNA interactions with observation of the movement and behavior of proteins over distances accessible by fluorescence microscopy. Generalized methods can be exploited to generate and optimize a variety of linear DNA substrates with plasmid DNA as a simple starting point using standard biochemical techniques. Here, we present protocols to produce high-quality plasmid-based 36-kb linear DNA substrates that support DNA replication by the Escherichia coli replisome and that contain chemical lesions at well-defined positions. These substrates can be used to visualize replisome-lesion encounters at the single-molecule level, providing mechanistic details of replisome stalling and dynamics occurring during replication rescue and restart.
Publisher: Elsevier
Date: 2016
DOI: 10.1016/BS.ENZ.2016.04.001
Abstract: DNA replication in Escherichia coli initiates at oriC, the origin of replication and proceeds bidirectionally, resulting in two replication forks that travel in opposite directions from the origin. Here, we focus on events at the replication fork. The replication machinery (or replisome), first assembled on both forks at oriC, contains the DnaB helicase for strand separation, and the DNA polymerase III holoenzyme (Pol III HE) for DNA synthesis. DnaB interacts transiently with the DnaG primase for RNA priming on both strands. The Pol III HE is made up of three subassemblies: (i) the αɛθ core polymerase complex that is present in two (or three) copies to simultaneously copy both DNA strands, (ii) the β2 sliding cl that interacts with the core polymerase to ensure its processivity, and (iii) the seven-subunit cl loader complex that loads β2 onto primer-template junctions and interacts with the α polymerase subunit of the core and the DnaB helicase to organize the two (or three) core polymerases. Here, we review the structures of the enzymatic components of replisomes, and the protein-protein and protein-DNA interactions that ensure they remain intact while undergoing substantial dynamic changes as they function to copy both the leading and lagging strands simultaneously during coordinated replication.
Publisher: Elsevier BV
Date: 2020
Publisher: Oxford University Press (OUP)
Date: 15-02-2019
DOI: 10.1093/NAR/GKZ090
Publisher: eLife Sciences Publications, Ltd
Date: 15-04-2017
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2024
End Date: 12-2026
Amount: $455,237.00
Funder: Australian Research Council
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