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

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.

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.

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.