Termination of DNA replication - a novel threat to genomic stability and cell cycle control
Lead Research Organisation:
Brunel University London
Department Name: Life Sciences
Abstract
Cancer is rapidly becoming one of the most common causes of death in human populations, and more than one in three individuals will suffer from it within their lifetime. A hallmark of cancer is the uncontrolled growth of cells, which is normally prevented by a network of control mechanisms that restricts the number of divisions normal cells can do. Cells that are damaged or have reached a certain age are prevented from any further divisions to remove the potential for them becoming cancerous. However, this continuous removal of old and damaged cells comes at a cost. While it avoids the formation of cancerous cells, it also increasingly impedes the regeneration of tissues, which is thought to be a major factor contributing to the ageing process of an individual. Thus, oncogenesis and ageing are opposite but tightly linked forces.
My interest in cancer genetics and ageing was one of the key motivations for studying molecular biology and for most of my research up to now. Initially, the medical aspects of cancer were my main interest. I spent several months in a human genetics laboratory where I worked directly with cancerous tissue. However, this work, although interesting, left me rather unsatisfied. Little was known about the details of cancer development in human cells and the studies I was doing felt rather like shots in the dark. Shortly afterwards I did a course in a lab that was interested in genomic stability in baker's yeast. Here I experienced that the understanding of the molecular details of DNA metabolism in yeast was far advanced in comparison to human cells. I was able to do simple experiments that allowed me to understand why the genetic information became unstable. In humans this lack of genomic stability is one of the key stages that results in transformation of a normal cell into a cancer cell, as it corrodes the complex network of quality control systems that maintains genomic integrity and ensures that cells only divide once it is safe to do so.
Motivated by this key feature of the biology of cancer, I went on to investigate how a specific yeast protein called Mph1, which was newly discovered at the time, helps to maintain the genetic information. These studies formed the basis of my Diploma and subsequent PhD training. A similar protein has been identified in humans. It is called FancM since its absence causes Fanconi anemia, a genetic disease associated with a much-elevated risk of cancer. It was exciting to see how investigations of a yeast protein can be of interest for human geneticists.
After completing my PhD I decided to study a protein called RecG, a DNA processing enzyme found in almost all species of bacteria. It was thought that RecG, Mph1 and FancM might all have a similar function in DNA metabolism. However, my studies revealed that RecG has an important and previously unknown function. It limits a major cause of genomic instability, one associated with events necessary for the orderly completion of chromosome replication, a fundamental requirement in all organisms. This trigger of genomic instability was unexpected and I am excited by the prospect of dissecting the molecular details. Ultimately, I aim to understand how it might contribute to the development of cancer and ageing. However, my initial studies will continue to exploit more tractable bacterial models so as to build some basic understanding before progressing to the more complex systems operating in higher organisms. As such my studies will also shed light on aspects of genomic instability that enable bacterial pathogens to overcome host defences and to acquire resistance to antibiotics.
My interest in cancer genetics and ageing was one of the key motivations for studying molecular biology and for most of my research up to now. Initially, the medical aspects of cancer were my main interest. I spent several months in a human genetics laboratory where I worked directly with cancerous tissue. However, this work, although interesting, left me rather unsatisfied. Little was known about the details of cancer development in human cells and the studies I was doing felt rather like shots in the dark. Shortly afterwards I did a course in a lab that was interested in genomic stability in baker's yeast. Here I experienced that the understanding of the molecular details of DNA metabolism in yeast was far advanced in comparison to human cells. I was able to do simple experiments that allowed me to understand why the genetic information became unstable. In humans this lack of genomic stability is one of the key stages that results in transformation of a normal cell into a cancer cell, as it corrodes the complex network of quality control systems that maintains genomic integrity and ensures that cells only divide once it is safe to do so.
Motivated by this key feature of the biology of cancer, I went on to investigate how a specific yeast protein called Mph1, which was newly discovered at the time, helps to maintain the genetic information. These studies formed the basis of my Diploma and subsequent PhD training. A similar protein has been identified in humans. It is called FancM since its absence causes Fanconi anemia, a genetic disease associated with a much-elevated risk of cancer. It was exciting to see how investigations of a yeast protein can be of interest for human geneticists.
After completing my PhD I decided to study a protein called RecG, a DNA processing enzyme found in almost all species of bacteria. It was thought that RecG, Mph1 and FancM might all have a similar function in DNA metabolism. However, my studies revealed that RecG has an important and previously unknown function. It limits a major cause of genomic instability, one associated with events necessary for the orderly completion of chromosome replication, a fundamental requirement in all organisms. This trigger of genomic instability was unexpected and I am excited by the prospect of dissecting the molecular details. Ultimately, I aim to understand how it might contribute to the development of cancer and ageing. However, my initial studies will continue to exploit more tractable bacterial models so as to build some basic understanding before progressing to the more complex systems operating in higher organisms. As such my studies will also shed light on aspects of genomic instability that enable bacterial pathogens to overcome host defences and to acquire resistance to antibiotics.
Technical Summary
What happens if two replication complexes collide? Termination of DNA synthesis occurs hundreds if not thousands of times each cell cycle in eukaryotic cells but just once in bacteria. I have demonstrated that in Escherichia coli fork collisions are associated with persistent hyper-replication of DNA, chromosome segregation defects and genomic instability, features typical for many cancer cells. This becomes apparent in strains lacking the dsDNA translocase RecG, which provides one major countermeasure by eliminating a substrate generated during fork collision that can allow re-loading of the replisome. The identification of termination zones in yeast (Fachinetti et al. 2010, Mol Cell 39:595-605) suggests that fork collisions may pose a similar threat in eukaryotic cells.
I will identify the intermediates resulting from fork collisions and investigate the systems that are normally processing these intermediates in E. coli. The distinct location of termination events in bacteria makes this approach quite feasible. My studies will establish whether replication fork collisions can cause genomic instability, especially in the absence of the systems that normally limit the pathologies associated with termination.
I will then extend my studies into eukaryotic models. I will investigate whether replication in human mitochondria, which has similarities to bacterial replication, suffers from similar problems. In the long term I will establish whether the hundreds of fork collisions in eukaryotic cells contribute to genomic instability. The insight into the factors maintaining genomic integrity is much needed for our understanding of ageing as well as cancer formation, prevention and treatment. In addition, my results will also significantly improve our understanding of the genetic adaptability that enables pathogenic bacteria and viruses to evade host defences and acquire resistance to antibiotics.
I will identify the intermediates resulting from fork collisions and investigate the systems that are normally processing these intermediates in E. coli. The distinct location of termination events in bacteria makes this approach quite feasible. My studies will establish whether replication fork collisions can cause genomic instability, especially in the absence of the systems that normally limit the pathologies associated with termination.
I will then extend my studies into eukaryotic models. I will investigate whether replication in human mitochondria, which has similarities to bacterial replication, suffers from similar problems. In the long term I will establish whether the hundreds of fork collisions in eukaryotic cells contribute to genomic instability. The insight into the factors maintaining genomic integrity is much needed for our understanding of ageing as well as cancer formation, prevention and treatment. In addition, my results will also significantly improve our understanding of the genetic adaptability that enables pathogenic bacteria and viruses to evade host defences and acquire resistance to antibiotics.
Planned Impact
My research will provide fundamental insights into DNA replication and the mechanics of replication termination. Termination is associated intrinsically with DNA replication and is therefore a fundamental aspect of the cell cycle in all organisms. In addition, the implications of my research address fundamental questions of the evolution of chromosomal architecture and replication speed in prokaryotes and eukaryotes (see research proposal). My results are already cited in current scientific literature reviews. Since they are important for our general understanding of DNA replication and the cell cycle, they will be of importance for teaching and textbooks aimed at undergraduates.
My research has already demonstrated some of the pathological consequences termination can have on genomic stability and cell cycle progression in E. coli. It will be important to further investigate the processes that are involved at this stage of the cell cycle and to identify the factors and systems that are limiting genomic instability. As a longer term objective I aim to explore how termination influences genomic stability in other bacteria, human mitochondria and eukaryotic cells. In human cells genomic instability is not only fueling senescence and ageing but it is also associated with an increased risk of cancer development. The detailed knowledge of the causes of genomic instability is important not only for the development of therapeutic agents needed for effective treatment of tumours, but also for the identification of markers that can be used for the diagnosis of genetic defects. Currently only very little research is carried out on replication termination and its link to genomic instability and my work will contribute towards strengthening the international competitiveness of the research on genomic stability, ageing and cancer carried out within the UK.
My studies of DNA replication and genome stability in bacteria will potentially have a direct impact on medical and biotechnological applications. Streptomycetes, which are the most important source of antibiotics for medical, veterinary and agricultural use, normally have a linear chromosome, which can circularise. This circularisation leads to a significant increase of chromosomal instability and my studies implicate that this instability is likely to be caused by replication fork collisions. A better understanding of the consequences and pathologies of fork collision events will therefore be of relevance for technical applications such as large scale culturing of Streptomycetes for production of antibiotics or other secondary metabolites of biological or chemical relevance. Furthermore, RecG helicase, which I have identified to be one of the key players in defusing potentially harmful replication fork collision intermediates, is involved in pilin antigenic variation in pathogens such as Neisseria gonorrhoeae. RecG is present in most bacterial species but has no known eukaryotic counterpart. Thus, it appears an attractive avenue for the development of drugs affecting the mechanisms developed to escape attacks by the host immune system. Thus, my studies will have relevance to medicine, agriculture and industry.
I will continue to make my data accessible by open access publications in scientific journals and by presentations at national and international conferences and workshops, which will allow me to discuss my findings with interested parties from the academic, medical and industrial communities. Furthermore, Brunel University has a unit dedicated to the dissemination and commercialisation of scientific research (Research Support and Development Office, RSDO), which facilitates communication with parties that are interested in commercial exploitation and I am already in contact to establish industrial collaborations for specific aspects of my work.
My research has already demonstrated some of the pathological consequences termination can have on genomic stability and cell cycle progression in E. coli. It will be important to further investigate the processes that are involved at this stage of the cell cycle and to identify the factors and systems that are limiting genomic instability. As a longer term objective I aim to explore how termination influences genomic stability in other bacteria, human mitochondria and eukaryotic cells. In human cells genomic instability is not only fueling senescence and ageing but it is also associated with an increased risk of cancer development. The detailed knowledge of the causes of genomic instability is important not only for the development of therapeutic agents needed for effective treatment of tumours, but also for the identification of markers that can be used for the diagnosis of genetic defects. Currently only very little research is carried out on replication termination and its link to genomic instability and my work will contribute towards strengthening the international competitiveness of the research on genomic stability, ageing and cancer carried out within the UK.
My studies of DNA replication and genome stability in bacteria will potentially have a direct impact on medical and biotechnological applications. Streptomycetes, which are the most important source of antibiotics for medical, veterinary and agricultural use, normally have a linear chromosome, which can circularise. This circularisation leads to a significant increase of chromosomal instability and my studies implicate that this instability is likely to be caused by replication fork collisions. A better understanding of the consequences and pathologies of fork collision events will therefore be of relevance for technical applications such as large scale culturing of Streptomycetes for production of antibiotics or other secondary metabolites of biological or chemical relevance. Furthermore, RecG helicase, which I have identified to be one of the key players in defusing potentially harmful replication fork collision intermediates, is involved in pilin antigenic variation in pathogens such as Neisseria gonorrhoeae. RecG is present in most bacterial species but has no known eukaryotic counterpart. Thus, it appears an attractive avenue for the development of drugs affecting the mechanisms developed to escape attacks by the host immune system. Thus, my studies will have relevance to medicine, agriculture and industry.
I will continue to make my data accessible by open access publications in scientific journals and by presentations at national and international conferences and workshops, which will allow me to discuss my findings with interested parties from the academic, medical and industrial communities. Furthermore, Brunel University has a unit dedicated to the dissemination and commercialisation of scientific research (Research Support and Development Office, RSDO), which facilitates communication with parties that are interested in commercial exploitation and I am already in contact to establish industrial collaborations for specific aspects of my work.
People |
ORCID iD |
Christian Rudolph (Principal Investigator) |
Publications
Dimude J
(2018)
Replication-transcription conflicts trigger extensive DNA degradation in Escherichia coli cells lacking RecBCD
in DNA Repair
Dimude JU
(2016)
Replication Termination: Containing Fork Fusion-Mediated Pathologies in Escherichia coli.
in Genes
Hawkins M
(2019)
Direct removal of RNA polymerase barriers to replication by accessory replicative helicases.
in Nucleic acids research
Ivanova D
(2015)
Shaping the landscape of the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic replication origin.
in Nucleic acids research
Lloyd RG
(2016)
25 years on and no end in sight: a perspective on the role of RecG protein.
in Current genetics
Midgley-Smith S
(2018)
Chromosomal over-replication in Escherichia coli recG cells is triggered by replication fork fusion and amplified if replichore symmetry is disturbed
in Nucleic Acids Research
Midgley-Smith SL
(2019)
A role for 3' exonucleases at the final stages of chromosome duplication in Escherichia coli.
in Nucleic acids research
Syeda AH
(2020)
Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics.
in Frontiers in microbiology
Title | It starts with a change |
Description | Poster created for the "Comic Sans for Cancer" competition |
Type Of Art | Artwork |
Year Produced | 2014 |
Impact | Raise awareness for how genomic instability is linked with cancer development |
URL | http://www.comicsanscancer.com |
Description | Objective 1 of the proposal is to investigate whether the recombination events associated with the fusion of replication forks can lead to genomic instability in bacteria. - In line with Objective 1a of the original proposal we have developed strains with tandem repeat cassettes in locations where replication forks collide as well as control locations. We have collected data sets and are in the process of completing a first stage with this particular experimental system. - In line with Objective 1b we have started to generate synthetic strains with additional replication origins and additional termination sites. These strains have generated a wealth of data, some of which are now published (Ivanova et al., 2015, Nucleic Acids Res. 43(16):7865-77). Our study has directly resulted in the generation of a set of strains with additional ectopic replication origins in different locations, allowing us to generate double and triple origin constructs with ectopic termination sites in a variety of places. We are currently writing another research paper to present our findings. In addition, we have further characterised the molecular details of replication fork fusion events. In a recent publication we were able to present data strongly suggesting that over-replication of the chromosome observed in the absence of RecG helicase is indeed triggered by fork fusion events, while over-replication in cells lacking RNase HI is triggered by a different mechanism, which clarifies the roles of these two proteins in cell metabolism (Dimude et al., 2015, mBio 6(6):e01294-15). In addition, we were able to demonstrate that in cells in which replication is initiated from an ectopic replication origin only, the consequences of replication fork fusion events in the termination area in the absence of RecG are so severe that the cells are inviable, an effect that can be efficiently suppressed if the replication fork trap in the termination area is inactivated. We have made important progress towards identifying the precise nature of this pathology. In addition, we were now able to demonstrate that over-replication in the absence of RecG can be triggered in ectopic locations if forks are forced to fuse in different chromosomal areas. A research manuscript will be submitted as a matter of days to Nucleic Acids Research. Furthermore we are investigating the roles of 3' exonucleases in fork fusion events, which we have identified as key players in defusing fork fusion intermediates (Rudolph et al., 2013, Nature 500(7464):608-11). We have made important progress with this part of the project and we are currently in the process of combining all data into a research paper which will be submitted within the next 3 months. The results of this study will help us to establish the molecular details of fork fusion events in vivo. - In line with Objective 1c we have established 2D DNA gel electrophoresis in the lab. However, we found that, while being able to demonstrate the presence of paused forks, we found very little signal for fork fusion intermediates, probably because they are short-lived and processed by a number of different proteins such as RecG and 3' exonucleases very rapidly. As 2D DNA gel electrophoresis is very time-consuming, we have at the moment suspended these experiments. Instead we have started with fluorescence microscopy studies following replisomes in living E. coli cells. We are currently measuring specific cell cycle parameters, which will allow us to determine the precise timing of when forks are going to fuse in relation to the initiation event at the replication origin. Objective 2 of the proposal is to investigate whether origin-independent synthesis can help to duplicate the bacterial chromosomes in times of genotoxic stress. - We have completed two studies showing that replication initiating in locations other than the origin leads to head-on collisions between replication fork and transcription complexes (Ivanova et al., 2015, Nucleic Acids Res. 43(16):7865-77; Dimude et al., 2015, mBio 6(6):e01294-15). Our data support the idea that the induced clashes between transcription and replication have a severe impact and, if unprocessed, can threaten the viability of the affected cells. Thus, while damage-induced forks are in theory capable of helping progression of ongoing synthesis to ensure survival of the cells affected, our data suggest that these forks also pose a danger, leading to the question of why and how damage induced synthesis is triggered. We will extend the fluorescence microscopy studies mentioned above to monitor locations and conditions that trigger such over-replication. We are currently working on the revision of a manuscript for another research paper demonstrating that the situation is much more complex, with replication-transcription clashes being only part of the story. Our data shed light on how the distinct features of bacterial chromosomes have evolved in which processes have the biggest impact on shaping the landscape of bacterial chromosomes. Objective 3 of the proposal is to investigate target proteins that might be involved in replication and termination of the human mitochondrial DNA. Key proteins will be expressed in E. coli cells with a termination defect and the efficiency of suppression of these proteins evaluated. The long term objective is to establish whether replication fork collisions might pose a problem in mitochondria, as replication has great similarities to bacterial replication. - We have successfully expressed human mitochondrial (hmt) RNase H1 in E. coli cells lacking RNase HI. We were able to show that the phenotype caused by the lack of RNase HI can indeed be partially complemented by hmt RNase H1. Furthermore, hmt RNase H1 was shown to play a role in mtDNA replication. We are now identifying further candidates that can be tested in a similar way. In addition, we have started to culture human fibroblasts to work directly with human mitochondria. Some mitochondrial DNA has been extracted and will be submitted for whole genome sequencing. These experiments are currently ongoing. |
Exploitation Route | In 2014 and 2015 I have given multiple oral and poster presentation as part of research seminars and conferences, which has greatly improved visibility of our work in termination (see relevant parts in the >>Common outputs<< section) and in 2016 my PhD student has presented a poster at one of the key conferences for DNA repair and homologous recombination. Furthermore, my postdoc has just been selected to present some of our findings at the Annual Conference of the Microbiology Society as part of the Prokaryotic Genetics and Genomics forum. In addition, we have published to literature reviews which highlight the importance of termination in the context of DNA replication and cell cycle progression (Lloyd & Rudolph 2015, Curr Genet. 62(4):827-840; Dimude et al. 2015 Genes (Basel) 7(8). pii: E40). Key findings of my work were published in 2013 in a letter in Nature, which was recently cited in a number of high profile journals by groups working on termination in bacteria as well as eukaryotic cells (e.g. Moreno et al. 2014, Science 346(6208):477-481; Maric et al. 2014, Science 346(6208):1253596; Dewar et al. 2015, Nature 525(7569):345-50; Wendel et al. 2014, PNAS doi: 10.1073/pnas.1415025111), highlighting the importance of the work undertaken. Finally, in summer 2016 we have organised and hosted the Symposium "DNA integration, replication dynamics and replication termination in E. coli", a specific platform to probe conceptual interactions between the fields of DNA replication, replication termination, Cas-CRISPR and transposon integration. This focussed meeting was very successful and has led to new defined collaborations aiming to specifically investigate the topic between termination and Cas-CRISPR spacer acquisition as well as transposon integration. Recently my team members were also more and more involved in presenting work from the lab in the form of posters and oral presentations. As indicated in other sections we have now develobed a laboratory webpage, which will provide a platform to make our results accessible to specialists as well as lay people and we are working on improving the accessibility of the content. In addtion, we are aiming now to expand into social networks such as facebook and twitter to increase our visibility as much as possible. |
Sectors | Education Healthcare Pharmaceuticals and Medical Biotechnology |
URL | http://www.rudolphlab.com |
Description | Staff training Both co-workers employed on the grant are highly trained and continuous training is provided. In addition to being familiar with all aspects of a modern molecular biology lab, they have received specific training in a number of highly specialised procedures, such as using an Amnis ImageStream Mark II for high-resolution in-flow microscopy, visualisation of protein and chromosome dynamics in vivo via fluorescence microscopy and specialised gel electrophoresis techniques, such as pulsed-field gel electrophoresis and 2D-DNA electrophoresis. Both have regularly presented in lab meetings and have successfully presented their findings at national and international research conferences, which will greatly enhance their communication skills, skills that are extremely important for being successful on the current national and international job market. My technician Sarah has started studies towards her PhD in the time of this grant and by now she has completed her PhD - she passed her viva in December 2018. She was successful in securing a position as postdoctoral research fellow in the Queen Mary Blizzard Institute. In addition, within the current grant period we have trained a number of undergraduate students to a very high level. Two of these have provided research data of such quality that they are joint first authors of a high-profile research paper (Ivanova, Taylor et al. 2015, Nucleic Acids Res. 43(16):7865-77). One of these two students graduated in summer 2015 and, even before graduation, was offered a job by PhlexGlobal. However, she was head-hunted by Parexel within a couple of months and is working for them now, demonstrating how the training provided improves the skills repertoire, allowing students trained in my lab to significantly contribute towards the wider economy of the UK. A second student was a named author on the above paper and is now doing her PhD at Cambridge University. She has been awarded the only VC award studentship of the University to finance her PhD work. A third student has greatly contributed to a current study and again her work was of such high quality that she is a co-author both on a review and a research paper (Dimude et al. 2016, Genes (Basel) 7(8), pii: E40; Dimude et al. 2018, Genes (Basel) 9(8), pii: E376). After doing a MSc degree she now has started studies towards her PhD at Heidelberg University in Germany. In addition, my lab has provided school students with a hands-on experience in molecular cloning and bacterial genetics, ranging from single students visiting the lab to whole classes gaining some practical experience. One such interaction is described below. http://www.brunel.ac.uk/news-and-events/news/news-items/ne_414554 These events regularly result in students being recruited to study STEM subjects at a University level, resulting not only in them gaining specialist knowledge such as Biomedical Sciences, but also contributing to the wider economy of the UK. Internet As detailed in our Pathways to Impact document we have developed a lab-specific website to make our research accessible to the interested public: www.rudolphlab.com We are aiming to continuously improve the content over the next few months to make our research results accessible to specialists and lay people alike. We will present our most recent research results on this platform and we are aiming now to expand into other social networks such as Facebook and Twitter to attract the widest readership possible. |
First Year Of Impact | 2017 |
Sector | Education,Healthcare,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal |
Title | Chromosomal over-replication in cells lacking 3' and 5' exonucleases |
Description | Replication profiles generated via Deep Sequencing analysing chromosomal over-replication in E. coli cells lacking the 3' exonucleases ExoI, ExoVII and SbcCD as well as the 5' exonuclease RecJ. |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Raw data are available for other researchers to scrutinise results published and to use the available data for mining in different areas of research, thereby driving forward current research projects. |
URL | http://www.ebi.ac.uk/ena/data/view/PRJEB28600 |
Title | DNA degradation in the termination area of cells lacking RecBCD |
Description | Bacterial chromosome duplication is initiated at a single origin (oriC). Two forks are assembled and proceed in opposite directions with high speed and processivity until they fuse and terminate in a specialised area opposite to oriC. Proceeding forks are often blocked by tightly-bound protein-DNA complexes, topological strain or various DNA lesions. In Escherichia coli the RecBCD protein complex is a key player in the processing of double-stranded DNA (dsDNA) ends. It has important roles in the repair of dsDNA breaks and the restart of forks stalled at sites of replication-transcription conflicts. In addition, ?recB cells show substantial amounts of DNA degradation in the termination area. In this study we show that head-on encounters of replication and transcription at a highly-transcribed rrn operon expose fork structures to degradation by nucleases such as SbcCD. SbcCD is also mostly responsible for the degradation in the termination area of ?recB cells. However, additional processes exacerbate degradation specifically in this location. Replication profiles from ?recB cells in which the chromosome is linearized at two different locations highlight that the location of replication termination can have some impact on the degradation observed. Our data improve our understanding of the role of RecBCD at sites of replication-transcription conflicts as well as the final stages of chromosome duplication. However, they also highlight that current models are insufficient and cannot explain all the molecular details in cells lacking RecBCD. |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | No notable impact yet, but the paper was already several times cited in the research literature, highlighting the academic impact. |
URL | http://www.ebi.ac.uk/ena/data/view/PRJEB27616 |
Title | DNA replication dynamics in Escherichia coli strains with an increasing number of ectopic replication origins |
Description | Following on from our oriZ work (Ivanova et al. 2015, Nucleic Acids Res.43(16):7865-77) we have generated strains with an additional ectopic replication origin termed oriX in the replichore opposite to the locatio of oriZ. Replication parameters were analysed in a way similar to our oriZ work, but we also managed to generate a strain background that carried 2 ectopic origins simultaneously, oriC+ oriX oriZ. Replication profiles were generated via Next Generation Sequencing and all Sequencing data will be made publically accessible via the European Nucleotide Archive. |
Type Of Material | Database/Collection of data |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | The datasets will allow researchers interested in replication dynamics and chromosome evolution in bacteria to study replication dynamics in cells with a variety of combinations of two, a single ectopic, two ectopic or three replication origins. |
URL | http://www.ebi.ac.uk/ena/data/view/PRJEB19883 |
Title | DNA replication dynamics in double origin cells lacking Rep helicase |
Description | Bacterial genome duplication and transcription require simultaneous access to the same DNA template. Conflicts between the replisome and transcription machinery can lead to interruption of DNA replication and loss of genome stability. Pausing, stalling and backtracking of transcribing RNA polymerases add to this problem and present barriers to replisomes. Accessory helicases promote fork movement through nucleoprotein barriers and exist in viruses, bacteria and eukaryotes. Here we show that stalled E. coli transcription elongation complexes block reconstituted replisomes. This physiologically relevant block can be alleviated by the accessory helicase Rep or UvrD, resulting in the formation of full-length replication products. Accessory helicase action during replication-transcription collisions therefore promotes continued replication without leaving gaps in the DNA. In contrast, DinG does not promote replisome movement through stalled transcription complexes in vitro. However, our data demonstrate that DinG operates indirectly in vivo to reduce conflicts between replication and transcription. These results suggest that Rep and UvrD helicases operate on DNA at the replication fork whereas DinG helicase acts via a different mechanism. |
Type Of Material | Database/Collection of data |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | No immediate impact yet, but the related paper is already cited in the scientific literature. |
URL | http://www.ebi.ac.uk/ena/data/view/PRJEB20003 |
Title | Over-replication in double origin cells lacking RecG helicase |
Description | Replication profiles of cells with one native, one ectopic or two replication origins, showing that areas where replication forks fuse are associated with over-replication of the bacterial chromosome |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | The raw data are available for other researchers not only for scrutinising published results, but also for mining the existing data in other context, thereby driving active areas of research forwards. |
URL | http://www.ebi.ac.uk/ena/data/view/PRJEB25595 |
Title | Synthesis in Escherichia coli strains with two replication origins |
Description | Deep sequencing datasets used to generate high resolution marker frequency replication profiles in E. coli strains with an additional replication origin (oriZ) in an ectopic location. The datasets are deposited with the European Nucleotide Archive. |
Type Of Material | Database/Collection of data |
Provided To Others? | No |
Impact | The datasets will allow researchers interested in replication dynamics and chromosome evolution in bacteria to study replication dynamics in cells with either two replication origins or a single replication origin in an ectopic location. |
URL | http://www.ebi.ac.uk/ena/data/view/PRJEB9476 |
Title | Synthesis in bacterial cells lacking RNase HI |
Description | Deep sequencing datasets used to generate high resolution marker frequency replication profiles in E. coli strains lacking RNase HI. The datasets are deposited with the European Nucleotide Archive and will be made publicly available upon acceptance of the related publication. |
Type Of Material | Database/Collection of data |
Provided To Others? | No |
Impact | The datasets will allow researchers interested in replication dynamics and chromosome evolution in bacteria to study sites of origin-independent replication initiation triggered by the absence of RNase HI. |
Description | Bernard Hallet |
Organisation | Catholic University of Louvain |
Country | Belgium |
Sector | Academic/University |
PI Contribution | Prof. Hallet's group investigates the molecular mechanism of transposon integration into bacterial chromosome and has found links of transposon integration with DNA replication and specifically replication termination. We have provided his lab with a number of strain constructs which will allow him to probe for various aspects of replication and termination. We have organised a Symposium in August 2016 with a variety of groups participating to have an active scientific exchange about links between replication, termination and DNA integration mechanisms. |
Collaborator Contribution | Exchange of ideas and concepts. |
Impact | No outputs yet. |
Start Year | 2016 |
Description | Conrad Nieduszynski |
Organisation | University of Oxford |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Exchange of research ideas and concepts. |
Collaborator Contribution | Data analysis, exchange of research ideas and concepts. |
Impact | Rudolph CJ, Upton AL, Stockum A, Nieduszynski CA, Lloyd RG (2013). Avoiding chromosome pathology when replication forks collide. Nature 500(7464):608-11. |
Start Year | 2008 |
Description | David Sherratt |
Organisation | University of Oxford |
Department | Department of Biochemistry |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Exchange of ideas and research data. |
Collaborator Contribution | We are sharing research data that are going to be published together. |
Impact | Ivanova et al. 2015, Nucleic Acids Res. 43(16):7865-77. |
Start Year | 2013 |
Description | Ole Skovgaard |
Organisation | Roskilde University |
Country | Denmark |
Sector | Academic/University |
PI Contribution | Our lab has contributed a large number of datasets to some data from the Skovgaard lab, leading not only to a publication but also triggering a number of experiments that are currently developed and will result in future publications. |
Collaborator Contribution | Ole Skovgaard has significantly contributed towards our ongoing research by providing bacterial strains and other materials, but also by contributing towards manuscripts, some of which are already published and some of which are currently defeloped. |
Impact | Research papers: PMIDs 26160884; 30060465; 32351461 |
Start Year | 2014 |
Description | Peter McGlynn |
Organisation | University of York |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Our lab is working on in vivo system of characterising replication fork fusions in living cells. |
Collaborator Contribution | Peter McGlynn is an excellent biochemist, which complements my own cell biology expertise very well. He is actively working on characterising replication fork fusion in a in vitro system with purified components. Peter McGlynn retired in September 2018, and the collaboration was continued with Dr Michelle Hawkins, a postdoc who was trained in the McGlynn lab. |
Impact | Research papers: PMIDs 19941825; 20923786; 30869136 Joint BBSRC grant BB/N014995/1 "Precision to the very end: what happens when two replication forks converge during termination?" |
Start Year | 2007 |
Description | Renata Retkute |
Organisation | University of Warwick |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Renata Retkute is an expert for computer modelling of replication dynamics in yeast and bacteria. Her modelling expertise excellently complements my own cell biology knowledge. |
Collaborator Contribution | Analysis of experimental data, integration of experimental data into computer modelling scenarios, evaluation of modelling results, leading to refind experiments. |
Impact | Research papers: PMIDs 26160884; 26530381; 30060465 |
Start Year | 2013 |
Description | DNA integration, replication dynamics and replication termination in Escherichia coli |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I have organised the Symposium "DNA integration, replication dynamics and replication termination in E. coli" at Brunel University London in August 2016, which was attended by 4 research groups from the UK, one research group from Louvain-La-Neuve in Belgium, and a research group from the University of Zagreb in Croatia. All groups have a related but so far distinctly different research focus, but we have recently identified overlapping interests. The Symposium was aimed to develop the overlapping interests further into more formal collaborations between our groups. This approach was very successfull and has resulted in consolidating 2 direct collaborations. The Symposium was much appreciated by all participants and we are discussing whether we can repeat it in 2017 with an increased number of research groups. |
Year(s) Of Engagement Activity | 2016 |
Description | Invited Research Seminar NTU |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Other audiences |
Results and Impact | Invited seminar entitled "Colliding forks - structural parameters of the E. coli chromosome" at the School of Science and Technology, Nottingham Trent University |
Year(s) Of Engagement Activity | 2016 |
Description | Invited oral presentation at the 7th European Conference on Prokaryotic and Fungal Genomics, Göttingen, Germany |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited to present my talk >>Origins left, right and centre: increasing the number of initiation sites in the Escherichia coli chromosome<< in the group section "Synthetic Biology"at the 7th European Conference on Prokaryotic and Fungal Genomics, Göttingen, Germany. Some of the interactions that followed from this talk were with related industries, but also with PIs, PDRAs and PhD students. |
Year(s) Of Engagement Activity | 2017 |
URL | http://www.prokagenomics.org/ |
Description | Invited oral presentation at the Annual Conference of the Microbiology Society |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | My postdoc, Juachi U. Dimude, was invited to present our data relating to integrating additional replication origins into the Escherichia coli chromosoe at the Prokaryotic genetics and genomics forum as part of the Annual Conference of the Microbiology Society. |
Year(s) Of Engagement Activity | 2017 |
URL | http://www.microbiologysociety.org/events/annual-conferences/index.cfm/annual-conference-2017 |
Description | Invited research seminar at The University of Birmingham |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | As a direct result of my postdocs talk the Annual Meeting of the Society of Microbiology I was invited for a Research Seminar by Prof. Stephen Busby and Prof. David Grainger to present my work at the Institute of Microbiology and Infection, University of Birmingham in October 2017. I had very good interactions with the researchers and PIs at the IMI |
Year(s) Of Engagement Activity | 2017 |
Description | JUD Invited oral presentation at the Young Microbiologists Symposium 2017, John Innes Centre, Norwich, UK |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | My postdoc Dr Juachi U. Dimude was invited to present our work at the Young Microbiologists Symposium 2017, which took place in September 2017 at the John Innes Centre in Norwich. Her talk was entitled: >>A start in many places: Structural insights of Escherichia coli chromosome by introducing an increased number of replication origins<<. |
Year(s) Of Engagement Activity | 2017 |
Description | Poster presentation at Recombination conference (Alicante) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Sarah Midgley-Smith, one of my group members, presented her work as a poster with the title "Termination and origin-independent replication in Escherichia coli" at the Abcam conference "Mechanisms of Recombination", an internationally renowned conference that is attended by the main resesarchers working in the field of DNA replication, recombination and repair worldwide. |
Year(s) Of Engagement Activity | 2016 |
Description | Research Seminar University of Nottingham Spring 2018 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Other audiences |
Results and Impact | Invited Research Seminar at the School of Biosciences to introduce the recent findings of my lab. |
Year(s) Of Engagement Activity | 2018 |
Description | Research Seminar University of Warwick Summer 2018 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Undergraduate students |
Results and Impact | Research seminar at the School of Medicine, Warwick University, to describe the most recent research findings of our group. |
Year(s) Of Engagement Activity | 2018 |
Description | Research Seminar, University of Sussex, Brighton |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | The talk was well received and triggered a lot of questions and discussion. Visit has potentially triggered a collaboration. |
Year(s) Of Engagement Activity | 2014 |
Description | SLM Poster presentation at SoM Annual Meeting |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | My PhD student Sarah Midgley-Smith presented her poster on the role of 3' exonucleases in termination of DNA replication in Escherichia coli. The poster session was attended by all participants of the Annual Meeting of the Society of Microbiology and Sarah interacted with a number of researchers and research students. |
Year(s) Of Engagement Activity | 2017 |
URL | http://www.microbiologysociety.org/events/annual-conferences/index.cfm/annual-conference-2017 |
Description | Seminar Okinawa Institute of Science and Technology |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited to present the latest research of my group as a Research Seminar at the Okinawa Institute of Science and Technology, Graduate University, Onna, Okinawa, Japan. I was personally invited by Prof. Simone Pigolotti to give this research seminar. This meeting was attended both in person in via online connections by a large number of researchers from all over Japan and other locations. My talk sparked questions and discussions afterwards. |
Year(s) Of Engagement Activity | 2022 |
Description | Seminar Tokyo Metropolitan |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited to present the latest research of my group as a Research Seminar at the Institute of Medical Science, Tokyo Metropolitan University. I was personally invited by the Director of the Institute of Medical Science, Prof. Hisao Masai, to give this research seminar. This meeting was attended both in person in via online connections by a large number of researchers from all over Japan and other locations. My talk sparked questions and discussions afterwards. |
Year(s) Of Engagement Activity | 2022 |