Precision to the very end: what happens when two replication forks converge during termination?

Lead Research Organisation: Brunel University
Department Name: Life Sciences

Abstract

DNA encodes the information that provides the basis for all life. For cell division to take place the entire DNA of a cell has to be fully duplicated, ensuring that both the mother and the daughter cell can get one complete copy. In addition, the copy made has to be identical to the original. Any changes to the DNA can potentially be harmful, as they will alter how cells function, or even lead to cell death. On a single cell level, accuracy of the duplication process is normally so high that not a single error is made when the millions of DNA base pairs are copied. This extraordinary high level of accuracy is achieved by a network of different processes that regulate the duplication process and choreograph segregation of the two complete copies into mother and daughter. However, mutations in the DNA can inactivate this network of processes and it is a hallmark of cancer cells that they grow in an uncontrolled way.

We are studying the final stages of the duplication process. Replication of DNA is initiated at specific sites called origins of replication. Two complex machines termed replication forks are recruited to these origins. These replication forks are capable of copying the DNA with high precision. While doing so they move in opposite direction at very high speed until they meet another replication fork coming the opposite way. Our research has revealed that the collision of two fast moving replication forks has the potential to corrupt the DNA and introduce mutations. This cause of mutation is unexpected, because the fusion of two such replication forks is a fundamental process when DNA is copied, and we have identified a number of pathways that can prevent the harmful consequences of fork fusions.

Currently we are using the bacterium Escherichia coli to gain a better insight into the mechanics of such fork fusion events. In E. coli only one such fusion event occurs, as the entire DNA is copied by only two replication forks. In our own cells the duplication process is initiated at hundreds of origins, leading to hundreds of fusion events, making studies much more complex. The relative simplicity of fork fusions in E. coli will allow us to study these events at very high detail with many different tools. For example, we will use recently developed microscopy techniques which allow us to directly visualise and track single replication forks inside living cells to study what happens if forks fuse and to identify how these fusion events can cause corruption of the DNA. Our results will allow us to generate a model of how the duplication process of DNA is normally brought to an orderly completion, which will help us to understand this process in our own cells, and our work will reveal whether it might contribute to the development of cancer and ageing.

Technical Summary

All organisms need to replicate their chromosomes with high fidelity to ensure that the genetic information passed on to the next generation is sufficiently accurate. Chromosome duplication initiates at defined origins, with two replication forks proceeding in opposite directions. DNA replication terminates when a replication fork meets the end of a chromosome or another fork travelling in the opposite direction. We have demonstrated in Escherichia coli that fork fusion events, if not processed correctly, result in surprisingly severe consequences, such as persistent over-replication of the chromosome, increased recombination and chromosome segregation defects. Thus, for the accurate completion of genome duplication the fusion of two converging forks must be carefully controlled, a theme also emerging for the hundreds of fork fusion events in eukaryotic cells.

While we have identified some of the pathologies that arise if fork fusions are not processed correctly, our understanding of the molecular mechanics of fork fusion is still limited. Here we propose to use a combined in vivo and in vitro approach in E. coli to directly analyse the protein dynamics and the DNA intermediates arising at fusing forks. We will investigate how fork fusion intermediates are processed and what happens when this processing goes awry, and we will determine how termination is choreographed in the context of whole chromosome dynamics, segregation and cell division. These analyses will provide a detailed view of replication termination and how the incorrect processing of fork fusions can result in pathologies. Our data will form an important foundation for the understanding of how the hundreds of fork fusions in eukaryotic cells are achieved and how their processing contributes towards maintaining genomic stability. Insight into the factors maintaining genomic integrity is much needed for our understanding of cancer, ageing and many hereditary diseases.

Planned Impact

The described programme will provide fundamental insights into what happens when two complex and fast moving replication forks converge and finally fuse. The fusion of replication forks is a necessity of DNA replication and therefore a fundamental aspect of the cell cycle in all organisms. In addition, the implications of our research address fundamental questions of the evolution of chromosomal architecture and replication speed in pro- and eukaryotes. Our studies will shed light on the mechanisms that have evolved to deal with the intermediates arising as forks fuse to allow duplication of the entire chromosome with a fidelity sufficient to avoid significant corruption of the genomic information.

Our recent research in E. coli has demonstrated that fork fusions can result in pathological consequences such as extensive over-replication of the chromosome, increased recombination and problems with cell cycle progression. It will be important to establish a mechanistic basis of how fork fusions are processed to limit genomic instability, as mistakes made during DNA replication are crucial in the development of genetic disease and other mutation-driven problems such as cancer. Clinicians and scientists with interests in hereditary diseases will therefore benefit from our fundamental studies. The general mechanics of DNA duplication is similar in all living organisms and studies in bacterial model organisms have provided many paradigms for understanding these processes in more complex systems. Currently, little research is carried out on replication fork fusions and the potential impact on genomic stability and our results will significantly contribute towards strengthening the international competitiveness of the research on DNA replication and genomic stability carried out within the UK.

Our studies will also have impact on medical and biotechnological applications. Streptomycetes are an important sources for antibiotics. Their chromosome is normally linear, in contrast to many other bacterial species, but it can circularise. It was noted before that this circularisation results in a significant increase of chromosomal instability and it is very tempting to speculate that this instability is a consequence of aberrantly processed fork fusion intermediates. Our work therefore has the potential to 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, we have identified RecG helicase as one of the key players in defusing potentially harmful fork fusion intermediates. The combined deletion of recG and other genes involved in processing fork fusion intermediates is lethal in E. coli. RecG, while being present in most bacterial species, has no known counterpart in mammalian cells. Thus, the proposed work may be of long-term benefit to pharmaceutical applications aiming to develop new targets for inhibition of pathogenic bacteria. Thus, our studies will have relevance to medicine, agriculture and industry.

The proposed research will combine complex biochemical work, molecular genetics and cell biology studies as well as computer modelling approaches to whole genome replication, resulting in significant cross-disciplinary training of all scientists involved. This will strengthen the scientifically-literate workforce and therefore the international competitiveness of the UK. Understanding how healthy organisms maintain genomic stability and cell division, and what happens when these processes go awry, will have long-term benefits to the health and well-being of the UK population. In addition, all researchers of this project will be well-placed to engage with the public to communicate the links between genomes, mutation and the genetic basis of disease, topics of general interest to the public.

Publications


10 25 50
 
Description Objective 1 of the proposal is to characterise the DNA intermediates formed and the protein dynamics that occur at two converging replication forks.

- We have successfully established a number of different systems which allow tracking of active replisomes in living cells. By using fluorescently labelled replisome components we are able to follow progression of replication in living cells. We also are able to lable the two different replisomes in living cells in different colours, which will allow us to follow the dynamics of forks independently. In addition, we can use conventional a fluorescent repressor-operator system (FROS) to visualise key areas of the chromosome. In combination with fluorescently labelled replisome components we can track where in relation to a certain chromosomal area a replication fork is located. We are currently in the process of establishing all the necessary growth conditions that will allow us to predict with a degree of precision when replication forks fuse in the termination area, given a defined initiation time.

- In addition we are making excellent use of cells which contain an additional ectopic replication origin, which, as a consequence, generates ectopic fork fusion sites. We have established strain backgrounds with single ectopic replication origins in a number of different locations and we have even generated a strain in which three active origins, oriC and two ectopic origins, are present. We are already in the process of writing a manuscript to present some of our findings.

- Having an experimental system with origins in defined ectopic locations has proven an extremely powerful tool to investigate replication dynamics of forks moving in an orientation opposite to normal. We have started to investigate replication dynamics in these systems in the presence and absence of key accessory helicase, such as Rep and DinG. This work has proceeded towards its final stage and we will write our findings up as a research paper over the next few months.

Objective 3 of the proposal aims to address whether fork fusion events are mechanistically linked to other genetic elements required for the late stages of chromosome duplication, such as segregation and cell division, and how fork fusions proceed if they are physically separated from these processes.

- We have observed before that the absence of the RecBCD dsDNA end processing enzymes in E. coli leads to the degradation of DNA in the native termination area, but not in the ectopic termination area in strains with two active replication origins. We have started to investigate the precise nature of the degradation. Our data rule out that degradation is triggered by recombination, but an involvement of exonucleases was shown. We were able to show that the discrepancy of degradation is not dependent on the location of the ectopic replication origin but is observed in a variety of constructs with ectopic origins in different places. We will now proceed to investigate what precisely causes the discrepancies between the native and the ectopic termination area.
Exploitation Route It is too early in the award to make a prediction.
Sectors Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
URL http://www.rudolphlab.com
 
Description Staff training My co-worker employed on the grant was already employed on the previous grant and has been highly trained in all aspects of a modern molecular biology lab as well as training on more specialised equipment such as using an Amnis ImageStream Mark II for high-resolution in-flow microscopy and specialised gel electrophoresis techniques, such as pulsed-field gel electrophoresis and 2D-DNA electrophoresis. Currently I am training her specifically in advanced fluorescence microscopy techniques that will allow her to visualise protein and chromosome dynamics in living cells, one of the important techniques for this specific awards. She has regularly presented her results in lab meetings, both local and with collaborators, and she has just been selected to present her findings at the Annual Conference of the Microbiology Society in Edinburgh as part of the Prokaryotic Genetics and Genomics forum. These presentation activities will continue to enhance her communication skills, skills that are extremely important for being successful on the current national and international job market. In addition we continue to train undergraduate and postgraduate students at a very high level. Within the short period of this grant two of our undergraduate students have generated data of such high quality that they will be included in the list of authors for a research paper that we are currently preparing, a remarkable achievement for students at an undergraduate level. From previous experience we know that hese achievements will significantly improve their chances on the job market once they graduate. In addition, in March 2017 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. 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 as our work on the grant progresses 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 2016
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
 
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