Termination of DNA replication - a novel threat to genomic stability and cell cycle control

Lead Research Organisation: Brunel University
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.

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.

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.
 
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 are currently investigating the precise nature of this pathology.

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). These studies 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 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. 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.

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 will present their findings at 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. 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. Another student has greatly contributed to a current study and again her work was of such high quality that she is a co-author on a review published last year (Dimude et al. 2016, Genes (Basel) 7(8), pii: E40) and she will be included in a research paper that is currently in preparation. She is currently applying for PhD positions 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 2015
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal
 
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 We have developed experimental systems where we can introduce additional origins into defined ectopic locations of the chromosome. These systems have allowed us to investigate changes to replication dynamics in the absence of key proteins. In this particular study we have analysed the impact of lacking either Rep or DinG helicase on replication progressing in an orientation opposite to normal. 
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 either two replication origins in the presence and absence of accessory factors such as Rep and DinG. 
URL http://www.ebi.ac.uk/ena/data/view/PRJEB20003
 
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 Conrad Nieduszynski 
Organisation University of Oxford
Country United Kingdom of Great Britain & Northern Ireland (UK) 
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 Medical Sciences Division
Department Department of Biochemistry
Country United Kingdom of Great Britain & Northern Ireland (UK) 
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 Ed Bolt 
Organisation University of Nottingham
Department School of Physics and Astronomy
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution My lab is actively working on generating strains in which proteins of the Cas-CRISPR system can be localised in vivo.
Collaborator Contribution Ed Bolt's lab is working on the in vitro reconstitution of the Cas-CRISPR system in Escherichia coli. The collaboration will allow us to combine both in vivo and in vitro approaches to characterise the system in E. coli and other bacterial organisms.
Impact No outputs yet
Start Year 2016
 
Description Ole Skovgaard 
Organisation Roskilde University
Country Denmark, Kingdom of 
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 Ivanova et al. (2015), Nucleic Acids Res. 43(16):7865-77. doi: 10.1093/nar/gkv704
Start Year 2014
 
Description Peter McGlynn 
Organisation University of York
Country United Kingdom of Great Britain & Northern Ireland (UK) 
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, wich 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.
Impact So far we have co-authored a couple of publications: Guy et al. 2009, Mol Cell 36 (4), 654-666 Atkinson et al. 2011, Nucleic Acids Res 39 (3), 949-957 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 Nottingham
Country United Kingdom of Great Britain & Northern Ireland (UK) 
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 Renata has contributed to 2 publications that are published: Ivanova et al. 2015, Nucleic Acids Res. 18;43(16):7865-77 Dimude et al. 2015, MBio 6(6):e01294-15
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 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 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 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