Speeding and stuttering: analysing the dynamics of DNA replication at the single molecule level

a08e2332-8bd1-411f-b0f2-2deb33a49618

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BBSRC
BBSRC
BBSRC
BBSRC
BBSRC

£1,505,975

Feb. 4, 2013

Feb. 3, 2016

Every time a cell divides it must copy its genetic material so that each daughter cell receives a complete set of genes. Any mistakes made during this copying process can be disastrous as even a single mistake can have fatal consequences. Unfortunately many obstacles are present that can block this replication process. We have discovered that a major problem are the many proteins that coat the DNA and that are needed for the normal processes of packaging, reading and repairing the genetic material. These proteins, when bound to the DNA template, can block replication of the DNA and prevent completion of the copying process. Such blockage may also trigger mutations since rearrangements within the genetic material are induced when replication machines come to a halt. However, our recent work has shown that accessory motors help to clear proteins out of the path of the advancing replication machine, playing a vital role in normal DNA replication, and that physical interaction of these accessory motors with the replication machinery is critical for their normal function.

Understanding how replication machines move along protein-coated DNA is important. These patterns of movement will dictate the likelihood of completing genome duplication and the probability of mutations occurring. In this project we will discover how individual replication machines move along protein-coated DNA, and how accessory motors alter this movement, to establish how these machines react upon encountering protein barriers. Studying individual replication complexes is essential because the replication of different DNA molecules is not synchronised. Measuring a large number of molecules at the same time will not reveal differences in motor speed nor pausing of individual motors, but only an average rate of duplication. In addition, we know that wide variations in behaviour are seen when comparing individual complexes. Behaviours which are rare might be very important with respect to completion of accurate genome duplication. Rare events are much easier to detect by observing individual complexes. We will use an imaging technique that can detect single replication complexes as they duplicate DNA, allowing the responses of individual complexes to protein barriers to be monitored. We will also investigate how physical interaction of an accessory motor with the replication machinery helps the accessory motor to function. We hypothesise that this interaction stimulates relative movement between different parts of the enzyme, activating the motor function and helping to clear proteins ahead of the replication machine. We will use a technique that measures the distance between two different positions within a single molecule very accurately to determine whether interaction with the replication machinery induces movements within the accessory motor and whether this interaction stimulates motor activity.

Copying of DNA is highly conserved from bacteria to man and protein barriers are a problem for all organisms. Our work will identify how this universal problem affects the DNA copying process and the means by which cells reduce the impact of protein barriers on DNA copying. Understanding how cells overcome barriers to DNA copying will also help in the design of drugs that target the accessory enzymes needed for efficient copying. Such drugs could have potential applications as new antibiotics for the treatment of infectious diseases and chemotherapy agents for the treatment of cancer. The design of new synthetic organisms, for example to aid biofuel production, will also benefit from this project. New organisms must contain the genetic instructions to maintain the life of that cell and these genetic instructions must be able to be copied accurately to allow the organism to grow and divide. Understanding how cells copy their genetic material in the face of protein barriers will help with the design of such novel organisms.

Technical Abstract:
All organisms must copy their genetic material both efficiently and accurately but our recent work has highlighted the unavoidable problems replication forks face in duplicating DNA coated with proteins. The barriers that protein-DNA complexes present has led to the evolution of accessory motors that interact physically with the replisome and that clear proteins ahead of the fork, underpinning completion of chromosome duplication. Maintenance of replisome movement is also critical in the maintenance of genome stability as stalled forks promote recombination and genome rearrangements. However, we do not know how repeated collisions with nucleoprotein complexes affect replisome movement along DNA nor how accessory replicative helicases modulate this fork movement. This ignorance is compounded by the recently-observed heterogeneity in replisome behaviour. Individual replisomes may therefore respond to protein barriers in very different ways, an important consideration given that rare events can trigger genome instability. We will use single molecule imaging of DNA synthesis catalysed by reconstituted E. coli replisomes to determine the kinetics of fork movement and stalling upon repeated collisions with nucleoprotein complexes and also the modulation of fork movement by accessory replicative helicases. These experiments will determine not only the population-averaged response of replisomes to protein barriers but also the heterogeneity in fork movement, including rare events that may be critical in precipitating replicative catastrophe. We will also use single molecule FRET spectroscopy to determine the mechanism by which physical interaction with the replisome facilitates accessory replicative helicase function in E. coli. This work will establish the impact of protein barriers on replisome movement, the range of possible outcomes from such collisions and the mechanism by which accessory motors minimise this impact, emerging as a conserved theme of genome duplication.

Potential Impact:
For any organism to grow and divide the DNA inside its cells must be accurately copied. This ensures that, upon cell division, the two daughter cells each have an uncorrupted copy of the genetic blueprint. Any mistakes in this highly conserved process of DNA replication can result in mutations and cell death. This project aims to understand how replication machines move along DNA, how this movement can be disrupted and the mechanisms that cells possess to minimise this disruption. This work will provide underpinning knowledge for the development of new pharmaceuticals that target DNA replication to inhibit copying of the genetic material. Such therapies are very effective in inhibiting the growth of disease-causing organisms such as bacteria and are also important in the treatment of tumours since rapid, unregulated cell division is a hallmark of cancer cells. Our proposed research may therefore provide the pharmaceutical industry with new leads for the development of novel anti-microbial and anti-cancer agents. This project will also provide increased understanding of how mutations arise when DNA replication is disrupted. This information will benefit clinicians by enhancing our knowledge about the links between DNA replication and the formation of mutations, providing opportunities for the development of new therapeutic and diagnostic tools for genetic diseases and cancer.

The field of synthetic biology will also benefit from this proposed research. Synthetic biology aims to generate partially or wholly synthetic cells optimised for use in energy production, chemical synthesis and other environmentally and economically important processes. All such lifeforms must copy their DNA accurately so that they can divide and produce viable daughter cells. Our programme will provide insight into what must be included in synthetic lifeforms to ensure accurate copying of their genetic blueprints.

The public will be the ultimate beneficiaries of this work. Results from our experiments will provide potential new avenues for the development of pharmaceuticals related to human, animal and crop health whilst enhanced design of synthetic cells has the potential to contribute new solutions to major environmental challenges. Thus our work will contribute to the health and well-being of the population and also enhancement of the UK economy. This research will also make a significant contribution to the provision of a scientifically-literate workforce and so will enhance the economic competitiveness of the UK. The project is interdisciplinary, employing both biologists and physical scientists to analyse the complex biological machines that copy DNA. Researchers employed on this project will therefore receive excellent training in a wide range of techniques. Moreover, this project focuses on genes and genomes, cancer and the basis of genetic disease, all topics of interest to the general public. Thus researchers trained during this project will be well-placed to discuss these issues at public engagement events.