About us
Overview
© RudolphLAB, 2026
Our research has been funded by The Leverhulme Trust, the Royal Society and the BBSRC. Our work is currently supported by the MRC.
Research
© RudolphLAB, 2026
Our lab uses Escherichia coli as a model system to study how cells achieve this level of accuracy. Like many bacteria, E. coli has a single circular chromosome, with replication initiated at a single defined origin (oriC). Two replisomes assemble at the origin and travel in opposite directions around the chromosome, each copying one half at high speed and precision until they converge. The relative simplicity of this system, combined with the powerful genetic and molecular tools available, allows us to dissect processes that are far harder to study in eukaryotic cells, where replication is initiated at hundreds of origins simultaneously.
Cell division cycle of a bacterium such as E. coli. Cells harbour a single circular chromosome which contains a single replication origin called oriC. Two replication forks are recruited and duplicate the chromosome in opposite directions, thereby dividing the chromosome into a clockwise (red) and counterclockwise (blue) half or replichore. Once replication is complete the two chromosomes can be segregated into the two daughter cells.
© RudolphLAB, 2026
Our research currently spans four connected projects, each addressing a different facet of how genome integrity is maintained.
Replication termination and genomic stability
Schematic illustrating the complexity of two replication forks converging opposite the origin. In order to complete chromosomal replication the DNA between the replisomes has to be fully replicated.
© RudolphLAB, 2026
Our long-standing interest is in replication termination – the final stage of DNA replication, when two converging replication forks meet and fuse. Although fork fusion is a fundamental and unavoidable part of completing genome duplication, our research has shown that it is far from a passive event. The collision of two fast-moving replisomes has the potential to corrupt the genome by provoking unwanted recombination and aberrant processing of the DNA at the fusion site, and we have identified a number of pathways that prevent these harmful consequences. Key among these are the RecG helicase/translocase and a set of 3' exonucleases, which together process fork fusion intermediates and suppress termination-induced over-replication. Because E. coli completes replication with a single fusion event, rather than the hundreds that occur simultaneously in human cells, it provides an unmatched system for studying these events in detail. The insights we generate here are directly relevant to understanding how termination is normally brought to an orderly completion in eukaryotic cells, and how its failure might contribute to genome instability, ageing, and disease. Our cell biology work is complemented by in vitro work from Dr Michelle Hawkins' group (University of York).
Replication-transcription conflicts and replication restart
Replication forks frequently encounter obstacles as they travel along the chromosome, and one of the most significant is the transcription machinery operating on the same DNA template. Collisions between replisomes and RNA polymerases can stall the replication fork, and if not resolved correctly, these events can lead to mutation, chromosomal rearrangements and even cell death. Cells have therefore evolved an array of replication restart pathways that recognise stalled or broken forks and reload the replisome to resume DNA synthesis. We are investigating how these conflicts are resolved at the molecular level, how restart pathways are coordinated with ongoing transcription, and what determines whether a fork is rescued faithfully or processed in a way that compromises genome integrity. Because replication-transcription conflicts are a universal feature of all living cells, the principles we uncover in E. coli speak directly to processes that are central to genome maintenance across all domains of life. This project is a joint endeavour with Prof. Mark Leake (University of York), whose group provides single-molecule data that complements our genetic and cell biological approaches.
CRISPR–Cas systems and DNA repair
Expression of fluorescently tagged Cas1 reveals the localisation of functional Cas1-Cas2 complexes in living E. coli cells
© RudolphLAB, 2026
CRISPR–Cas systems are best known as bacterial adaptive immunity, providing protection against invading phages and plasmids. However, it is becoming increasingly clear that Cas proteins also intersect with the cell's own DNA replication and repair machinery in ways that are not yet well understood. Several Cas proteins exhibit nuclease and helicase activities that are reminiscent of those used in housekeeping repair pathways, raising the question of how these systems are coordinated within the cell. Disentangling the immunity-related and repair-related roles of Cas proteins is essential if we are to understand how bacteria balance defence against external threats with maintenance of their own genome. We contribute live-cell microscopy to a project led by Prof. Ed Bolt (University of Nottingham), working alongside Prof. Ivana Ivančić Baće (University of Zagreb), who provides genetic analyses. Together we are dissecting how Cas proteins localise, interact and function alongside replication and repair pathways in living cells.
Artificial sweeteners and bacterial physiology
E. coli cells treated for 60 min with the artificial sweetener saccharin show clear signs of severe stress. Dead cells can be easily distinguished by their distinctly lighter colour.
© RudolphLAB, 2026
More recently, we have begun investigating how artificial sweeteners – including acesulfame K and saccharin – affect bacterial cells and how this compares to treatment of cells with certain antibiotics. Artificial sweeteners are consumed in vast quantities worldwide and are widely assumed to be biologically inert, passing through the body without significant interaction. Our work, however, has shown that they exert substantial effects on bacteria: they disrupt DNA replication, compromise membrane integrity and ultimately they can promote cell death. These findings raise important questions about the impact of artificial sweeteners on the bacterial communities that inhabit the human gut, as well as their potential consequences for environmental microbial populations exposed to sweeteners through wastewater. Even more importantly, it might be possible to use these sweeteners to re-sensitise resistant bacterial pathogens to commonly used antibiotics and to potentially use them directly as antimicrobials themselves. Understanding the cellular mechanisms underlying these effects is a first step towards evaluating their broader biological significance. This project is led by Prof. Ronan McCarthy (University of Southampton), with our group contributing microscopy to characterise the cellular effects on replication and membrane organisation.
In order to measure and visualise different aspects of DNA replication, DNA repair and cell physiology we use a wide variety of tools and techniques.
Genetics
Example of the synthetic lethality screen. (A) shows cells in which the gene encoded on the pRC7 is not needed. Segregation of blue and white colonies is observed and blue colonies contain white sectors, demonstrating the rapid loss of the covering plasmid. (B) shows a synthetically sick interaction of two genes of interest. Cells contain a deletion of two genes, which renders the cells sick and very slow growing. Cells retaining the covering plasmid show rapid growth into unsectored blue colonies, as one of the two deletions is masked, while cells having lost the plasmid show very small white colonies, demonstrating that they are viable but very slow growing. (C) shows a synthetically lethal interaction of a deletion of two genes. Cells which have lost the covering plasmid are entirely unable to grow and no white colonies are observed, while cells retaining the covering plasmid grow into unsectored blue colonies.
© RudolphLAB, 2026
We use traditional genetics approaches for strain constructions and phenotypic investigations, such as P1vir transduction, single-step gene disruption and many others. We routinely perform phenotypic analyses such as doubling time, sensitivity to damage induced by a variety of genotoxic agents, delay of cell division following DNA damage, growth in the absence of an active origin etc. In addition, a synthetic lethality assay is particularly informative to assess the genetic interactions of genes and point mutations. This assay employs the unstable pRC7 plasmid, which is rapidly lost, with cloned genes of interest to cover for chromosomal deletions. pRC7 carries a functional lac operon, and its loss can be revealed in a lac– mutant background on plates containing the beta-galactosidase indicator X-Gal by the formation of white colonies or white sectors (lac–) within blue colonies, depending on whether plasmid loss occurred before or after plating. This assay can efficiently highlight synthetically lethal but also synthetically sick genetic interactions.
Visualisation of protein and chromosome dynamics
Time lapse visualisation of replication fork dynamics and terminus segregation in E. coli. The terminus region of the chromosome (cyan; visualised by lac repressor-fluorescent fusion proteins bound to lac operators) co-localises with replication forks (red focus) as it is replicated (i). All forks are disassembled at the termination area (ii – the red focus disappears) prior to chromosome segregation (iii – two cyan labelled termini appear). New replication forks are subsequently assembled (iv – a red focus in each of the daughter cells).
© RudolphLAB, 2026
For cell biological visualisations we use a number of modern tools. Protein dynamics at replication forks in living cells can be visualised by fusing proteins of interest to fluorescent proteins. This allows us to study localisations within the cell, but also protein dynamics as complexes might be assembled and disassembled. In addition, chromosome dynamics can be visualised using fluorescent repressor-operator systems (FROS), allowing the visulisation of the location of defined chromosomal areas. If combined together, both systems provide a measure of when active replisomes reach defined areas of the chromosome, enabling us to specifically study the events that occur when replisomes reach the termination area of the chromosome.
Molecular Cell Biology
Visualisation of synthesis in cells in which oriC-firing is inhibited via a temperature-sensitive allele of the DnaA initiator protein (dnaA(ts)). Cells were shifted to restrictive temperature for 90 min, allowing all ongoing replication forks to complete synthesis while new forks at oriC cannot be established. Cells were then pulse-labelled with EdU for 15 min and, following the click-labelling reaction, visualised with an ImageStreamX Mark II. In dnaA(ts) single mutants no or very little synthesis is observed, while cells lacking RNase HI show robust level of synthesis in 75% of cells, providing an explanation why cells lacking RNase HI are capable of surviving without an active replication origin.
© RudolphLAB, 2026
The molecular biology of DNA replication can not only be measured but also visualised by the incorporation of nucleotide analogues into nascent DNA. Incorporation of BrdU with subsequent separation of NotI-cut fragments of the chromosome via pulsed-field gel electrophoresis allows the visualisation of chromosomal areas with ongoing DNA synthesis in a population of cells. However, we can also visualise ongoing synthesis on a single cell level. By pulse-labeling newly replicated DNA with 5-ethynyl-2-deoxyuridine (EdU) followed by a click chemistry reaction and high-resolution microscopy in flow with an Amnis ImageStreamX Mark II, which combines the power of FACS analysis with single cell visualisation, we can quantify the amount of label while simultaneously imaging the location of where labelling is taking place (Dimude et al., 2015).
Replication profiles at a high resolution
Replication profiles in E. coli cells in the presence (i) and absence (ii) of RecG helicase/tranlocase. Profiling of exponential-phase cells normalized against a stationary wild type culture are shown. The genome-wide high-resolution marker frequency analysis reveals high level of over-replication in the termination area of the chromosome in cells lacking RecG.
© RudolphLAB, 2026
To visualise copy number variations of defined chromosomal areas, a variety of tools are used. qPCR is suitable to analyse relative abundance of defined chromosomal areas. To investigate whole chromosome replication dynamics, high-resolution replication profiles generated via Deep Sequencing can be used. This marker frequency analysis measures the duplication of each part of the genome as an increase in copy number following replication. It can very efficiently reveal aberrant processing of replication intermediates via under- and over-representation of specific DNA sequences. For example, it has allowed us to demonstrate that cells lacking RecG helicase/translocase show significant levels of over-replication specifically in the termination area of the chromosome. Our experiments have identified RecG, together with 3' exonucleases, as key proteins required for the processing of fork fusion intermediates to prevent termination-induced over-replication.
Rudolph BLOG
Kirby CMS – or a tale of two support teams
Two software companies. Two very different ideas of customer service.
Older articles
People
Lab members
Dr. Christian Rudolph
Helen Blakes
Undergraduate and MSc students
Amin Hashemloo
Iren Grigoryan
Previous lab members
Dominika Krawiel
Stelinda Peros
Emma L. Dunbar
Daniel J. Goodall
Dr Juachi U. Dimude
Brian Prince-Lombard
Sabiha Karim
Mohammad Khalifa
Ewa Andrzejewska
Dominika Warecka
Vladyslava Borysenko
Anastasia Georgievskaya
Alexandra Gajdosova
Toni Taylor
Sarah L. Midgley-Smith
Monja Stein
Darja Ivanova
Dr. Amy L. Upton
Publications
Great stuff we wrote is listed below. For plain-language summaries, highlights, and broader context, you can also explore my publications on Kudos.
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Goodall D.J., Dimude J.U., Hashemloo M.A., Dunbar E.L., Grigoryan I., Upton A.L., Bolt E.L. and C.J. Rudolph (2026). Termination of DNA replication drives genomic instability via multiple mechanisms. Nucleic Acids Res. 54(2):gkaf1519. DOI: 10.1093/nar/gkaf1519.
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de Dios R., Gadar K., Proctor C.R., Maslova E., Han J., Soliman M.A.N., Krawiel D., Dunbar E.L., Singh B., Peros S., Killelea T., Warnke A.-L., Haugland M.M., Bolt E.L., Lentz C.S., Rudolph C.J. and R.R. McCarthy (2025). Saccharin disrupts bacterial cell envelope stability and interferes with DNA replication dynamics. EMBO Mol Med. DOI: 10.1038/s44321-025-00219-1 [Link]
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Killelea T., Kemm F.E., He L., Rudolph C.J. and E.L. Bolt (2024). Repurposing Proximity-Dependent Protein Labeling (BioID2) for Protein Interaction Mapping in E. coli. Methods Mol Biol. 2828, 87–106. doi: 10.1007/978-1-0716-4023-4_9 [Link]
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Goodall D.J., Warecka D., Hawkins M. and C.J. Rudolph (2023). Interplay between chromosomal architecture and termination of DNA replication in bacteria. Front. Microbiol 14. DOI: 10.3389/fmicb.2023.1180848 [Link]
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Killelea T., Dimude J.U., He L., Stewart A.L., Kemm F.E., Radovčić M., Ivančić-Baće I., Rudolph C.J. and E.L. Bolt (2023). Cas1-Cas2 physically and functionally interacts with DnaK to modulate CRISPR adaptation. Nucleic Acids Research 51 (13), 6914–6926. DOI: 10.1093/nar/gkad473 [Link]
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de Dios R., Proctor C.R., Maslova E., Dzalbe S., Rudolph C.J. and Ronan R. McCarthy (2023). Artificial sweeteners inhibit multidrug resistant pathogen growth and potentiate antibiotic activity. EMBO Mol. Med. 15(1):e16397. DOI: 10.15252/emmm.202216397. [Link]
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Jameson K.H., Rudolph C.J. and M. Hawkins (2021). Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA. J Biol Chem. 297 (6), 101409. doi: 10.1016/j.jbc.2021.101409
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Goodall D.J., Jameson K.H., Hawkins M. and C.J. Rudolph (2021). A Fork Trap in the Chromosomal Termination Area Is Highly Conserved across All Escherichia coli Phylogenetic Groups. Int. J. Mol. Sci. 22 (15), 7928. DOI: 10.3390/ijms22157928.
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Midgley-Smith, S.L.; Dimude, J.U.; Taylor, T.; Forrester, N.M.; Upton, A.L.; Lloyed, R.G. and C. J. Rudolph (2018). Chromosomal over-replication in Escherichia coli recG cells is triggered by replication fork fusion and amplified if replichore symmetry is disturbed. Nucleic Acids Res. 46(15):7701-7715.
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Rudolph, C.J.; Upton, A.L.; Stockum, A.; Nieduszynski, C.A. and R.G. Lloyd (2013). Avoiding chromosome pathology when replication forks collide. Nature 500 (7464), 608–11.
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Syeda A.H., Dimude J.U., Skovgaard O. and C.J. Rudolph (2020). Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics. Front. Microbiol., 15 April 2020. doi: 10.3389/fmicb.2020.00534
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Hawkins M., Dimude J.U., Howard J.A.L., Smith A.J., Dillingham M.S., Savery N.J., Rudolph C.J. and P. McGlynn (2019). Direct removal of RNA polymerase barriers to replication by accessory replicative helicases. Nucleic Acids Res 47(10):5100–5113.
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Midgley-Smith, S.L., Dimude, J.U. and C.J. Rudolph (2018). A role for 3' exonucleases at the final stages of chromosome duplication in Escherichia coli. Nucleic Acids Res. 47(4):1847–1860
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Rudolph C.J., Corocher T.-A., Grainge I. and I.G. Duggin (2019). Termination of DNA Replication in Prokaryotes. Wiley electronic Library of Science (eLS). DOI: 10.1002/9780470015902.a0001056.pub3
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Dimude, J.U., Midgley-Smith, S.L. and C.J. Rudolph (2018). Replication-transcription conflicts trigger extensive DNA degradation in Escherichia coli cells lacking RecBCD. DNA Repair (Amst) 70:37–48.
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Dimude, J.U.; Stein, M.; Andrzejewska, E.E.; Khalifa, M.S.; Gajdosova, A.; Retkute, R.; Skovgaard, O. and C.J. Rudolph (2018). Origins Left, Right, and Centre: Increasing the Number of Initiation Sites in the Escherichia coli Chromosome. Genes 9 (8), pii: E376.
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Dimude, J.U.; Midgley-Smith, S.L.; Stein, M. and Rudolph, C.J. (2016). Replication Termination: Containing Fork Fusion-Mediated Pathologies in Escherichia coli. Genes 7(8), 40; doi:10.3390/genes7080040.
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Lloyd, R.G. and C.J. Rudolph (2016). 25 years on and no end in sight: a perspective on the role of RecG protein. Curr. Genet. 62(4):827–40. doi: 10.1007/s00294-016-0589-z
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Dimude, J.U.; Stockum, A; Midgley-Smith, S.L.; Upton, A.L.; Foster, H.A.; Khan, A; Saunders, N.J.; Retkute, R. and C.J. Rudolph (2015). The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin. mBio. 6(6). pii: e01294-15. doi: 10.1128/mBio.01294–15.
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Ivanova, D.; Taylor, T.; Smith, S.L.; Dimude, J.U.; Upton, A.L.; Mehrjouy, M.M.; Skovgaard, O.; Sherratt, D.J.; Retkute, R. and C.J. Rudolph (2015). Shaping the landscape of the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic replication origin. Nucleic Acids Res. 43(16):7865–77.
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Upton, A.L.; Grove, J.I.; Mahdi, A.A.; Briggs, G.S.; Milner, D.S.; Rudolph, C.J. and R.G. Lloyd (2014). Cellular location and activity of Escherichia coli RecG proteins shed light on the function of its structurally unresolved C-terminus. Nucleic Acids Res. 42 (9), 5702–14.
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Stockum, A.; Lloyd, R.G. and C.J. Rudolph (2012). On the viability of Escherichia coli cells lacking DNA topoisomerase I. BMC Microbiology 12:26.
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Atkinson, J.; Gupta, M.K.; Rudolph, C.J.; Bell, H.; Lloyd, R.G. and P. McGlynn (2011). Localisation of an accessory helicase at the replisome is critical in sustaining efficient genome duplication. Nucleic Acids Res 39 (3), 949–957.
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Ede C, Rudolph CJ, Lehmann S, Schürer KA, Kramer W. (2011). Budding yeast Mph1 promotes sister chromatid interactions by a mechanism involving strand invasion. DNA Repair 10 (1), 45–55
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Rudolph C.J., Mahdi A.A., Upton A.L. and R.G. Lloyd (2010). Single-strand DNA exonucleases protect Escherichia coli cells from the pathological consequences of unscheduled DNA replication. Genetics 186 (2), 473–492.
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Rudolph C.J., Upton A.L., Briggs, G.S. and R.G. Lloyd (2010). Is RecG a general guardian of the bacterial genome? DNA Repair 9 (3), 210–223.
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Guy C.P., Atkinson J., Gupta M.K., Mahdi A.A., Gwynn E.J., Rudolph C.J., Moon P.B., van Knippenberg I.C., Cadman C.J., Dillingham M.S., Lloyd R.G. and McGlynn P. (2009). Rep provides a second motor at the replisome to promote duplication of protein-bound DNA. Mol Cell 36 (4), 654–666.
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Rudolph C.J., Upton A.L. and R.G. Lloyd (2009). Replication fork collisions cause pathological chromosomal amplification in cells lacking RecG DNA translocase. Mol Microbiol 74 (4), 940–955.
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Rudolph C.J., Upton A.L., Harris L. and R.G. Lloyd (2009). Pathological replication in cells lacking RecG DNA translocase. Mol Microbiol 73 (3), 352–366.
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Rudolph C.J., Upton A.L. and R.G. Lloyd (2008). Maintaining replication fork integrity in UV-irradiated Escherichia coli cells. DNA Repair 7 (9), 1589–1602.
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Rudolph C.J., Upton A.L. and R.G. Lloyd (2007). Replication fork stalling and cell cycle arrest in UV-irradiated Escherichia coli. Genes Dev 21, 668–681.
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Rudolph C.J., Dhillon P., Moore T. and R.G. Lloyd (2007). Avoiding and resolving conflicts between DNA replication and transcription. DNA Repair 6 (7), 981–993.
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Rudolph C., Schürer, K.A. and W. Kramer (2007). Facing Stalled Replication Forks: The Intricacies of Doing the Right Thing, in Lankenau, D-H. (ed.) Genome Integrity: Facets and Perspectives. Springer, Vol. 1. pages 105–152. ISBN 13: 978-3-540-37531-9. DOI:10.1007/7050_003
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Schürer K.A., Rudolph C., Ulrich H.D. and W. Kramer (2004). Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair. Genetics 166 (4), 1673–86. doi: 10.1534/genetics.166.4.1673.
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Scheller J., Schürer A., Rudolph C., Hettwer S. and W. Kramer (2000). MPH1, a yeast gene encoding a DEAH protein, plays a role in protection of the genome from spontaneous and chemically induced damage. Genetics 155 (3), 1069–81. doi: 10.1093/genetics/155.3.1069.
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Contact us
Rudolph LAB
Department of Life Science
Heinz Wolff Building Room 247
Brunel University of London
Kingston Lane
Uxbridge, UB8 3PH
UK
We are always interested in motivated students with biology, genetics or biochemstry backgrounds and enquiries with CV are welcome. Please send them via email to Dr Christian Rudolph:
christian.rudolph@brunel.ac.uk.