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

Lead Research Organisation: University of York
Department Name: Biology

Abstract

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 Summary

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.

Planned 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.
 
Description The prevaling view of DNA replication until recently was that it continued from initiation through to termination with few if any breakdowns during this process. We now appreciate that replication forks stall frequently and that these breakdowns present major challenges to the completion of genome duplication and the maintenance of genome stability(1). Furthermore, we now know that proteins bound to the template DNA are a major cause of replication fork stalling(2). In most instances the replication machinery, driven by the primary replicative helicase, can displace any proteins bound to the template to allow continued unwinding and duplication of DNA. However, occasionally this displacement does not occur and the many protein-DNA complexes encountered by replisomes means that even a small probability of stalling at a single protein-DNA complex results in a major challenge to genome duplication(3,4). In spite of this importance, though, we still do not know how collisions with protein-DNA complexes alter replisome movement along DNA. Moreover, although we know that the primary replicative helicase is backed up by accessory helicases to aid fork movement through protein-DNA complexes, we do not know in what manner such accessory helicases alter replication fork movement along protein-bound DNA. This project seeks to analyse movement of replisomes directly along protein-coated DNA in vitro by visualising single E. coli replisomes. It also seeks to understand the impact of accessory helicases on replisome movement along protein-bound DNA. Furthermore, we know that the primary and accessory replicative helicases in E. coli, DnaB and Rep, interact physically and that this physical interaction results in cooperativity in DNA unwinding and the underpinning of fork movement along protein-bound DNA(5,6). However, we do not know how this interaction affects primary and accessory helicase function. This project also aims to determine how interaction between Rep and DnaB facilitates replisome movement.

We have established systems to visualise single replisomes in vitro. We have developed DNA templates on which we can reconstitute the E. coli replication machinery. These templates are so-called rolling circles which allow the replication machinery to traverse the template repeatedly, in effect travelling many hundreds of kilobases(7). Our reconstituted replisomes are fully functional on these templates, evinced by coupled leading and lagging strand synthesis with a processivity in excess of 100 kb. We have also engineered the templates to contain different types of model protein-DNA complex to act as barriers to replication and have combined this biochemical reconstitution with total internal reflection fluorescence (TIRF) microscopy to visualise single replisomes on these rolling circle templates. TIRF imaging has revealed replication products, some of which are several hundred kilobases long. These TIRF experiments are ongoing in Glasgow.

We have also probed how the interaction between DnaB and Rep facilitates fork movement. Domain motions within Rep are important for the ability of Rep to unwind DNA(8,9) and our initial hypothesis was that these domain motions might be influenced by the Rep-DnaB interaction, facilitating DNA unwinding by Rep. We surface immobilised wild type His-tagged Rep on PEG-coated slide surfaces bearing antibody specific to the His-tag. We have also shown that Rep immobilised in this manner is active by using DNA substrates labelled with donor-acceptor fluorophores arranged as a pair in opposing DNA strands such that DNA unwinding results in the disappearance of FRET. Given our ability to immobilise Rep on slides in an active state, we have now labelled Rep on pairs of engineered cysteine residues with donor and acceptor dyes positioned to provide direct FRET readouts of conformational motions. We have detected the "open" and "closed" conformations of wild type Rep formed by movement of the 2B subdomain(8,10). We have also analysed Rep conformation in the presence of DnaB. However, there appears to be no significant impact of addition of DnaB on the conformational status of Rep. These data indicate that interaction with DnaB does not affect switching between the open and closed states of Rep.

Absence of any effect of DnaB on Rep conformation implies that the observed cooperativity between Rep and DnaB in DNA unwinding(5,6) does not involve DnaB-induced alterations in the open versus closed state of Rep. This model suggests that the 2B subdomain itself may not be needed for cooperativity. Tim Lohman (Washington University School of Medicine, St Louis, Missouri) kindly supplied a rep overexpression plasmid that encodes a mutant Rep lacking this subdomain(11,12). We have found that Rep?2B displays cooperativity with DnaB when unwinding DNA substrates in ensemble reactions, as seen with wild type Rep. These data support our conclusion above that cooperativity in DNA unwinding via physical interaction of Rep with DnaB does not occur by altering the open versus closed status of Rep, with cooperativity being observed regardless of the presence or absence of the 2B subdomain. Our current working model is that tethering of Rep via a physical interaction with DnaB increases the effective local concentration of Rep within the vicinity of the DNA substrate, resulting in cooperative unwinding of DNA.

Rep?2B is activated for DNA unwinding as compared with the wild type enzyme(11). Combined with our discovery that Rep?2B also displays cooperativity with DnaB during DNA unwinding we expected Rep?2B to be at least as effective as the wild type enzyme in promoting movement of the replication machinery along protein-coated DNA. However, we found that Rep?2B failed to promote movement of the reconstituted E. coli replisome along protein-bound DNA. We then found that, in isolation, Rep?2B was severely defective in disrupting model protein-DNA complexes from both single-stranded and double-stranded DNA as compared with the wild type enzyme. These data were unexpected. They are also important as they indicate that DNA unwinding and nucleoprotein complex disruption can be uncoupled, demonstrating that protein displacement is an evolved function of helicases and not merely a consequence of translocation along DNA.

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8. Korolev, S., Hsieh, J., Gauss, G.H., Lohman, T.M. and Waksman, G. (1997) Cell, 90, 635.
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11. Brendza, K.M., Cheng, W., Fischer, C.J., Chesnik, M.A., Niedziela-Majka, A. and Lohman, T.M. (2005) Proc. Natl. Acad. Sci. U S A, 102, 10076.
12. Cheng, W., Brendza, K.M., Gauss, G.H., Korolev, S., Waksman, G. and Lohman, T.M. (2002) Proc. Natl. Acad. Sci. U S A, 99, 16006.
Exploitation Route Our findings are revealing how the DNA replication machinery can move efficiently along protein-coated DNA. Our work also demonstrates that movement of a molecular motor along DNA is insufficient in itself to push proteins off the template. Instead, helicases have evolved specific features to facilitate protein displacement from the DNA. This work has the potential to provide underpinning knowledge for the development of new antimicrobials that target DNA replication or many other aspects of bacterial nucleic acid metabolism that require helicase activities. Our findings might also inform healthcare professionals. It is now appreciated that blockage of replication machines is an important source of mutations. It is still unclear exactly what the relative importance of nucleoprotein barriers to replication is in terms of the development of genetic diseases and carcinogenesis but such blockage of replication forks is emerging as a common feature of genome duplication in all organisms. Understanding how organisms underpin replication of protein-bound DNA will therefore provide insight into the means by which organisms minimise associated mutations.

Our findings raise the question of exactly how helicases have evolved to displace proteins from DNA. Our work demonstrates that helicase translocation along DNA is insufficient for this displacement and so the mechanistic basis of promoting protein displacement needs to be probed in order to understand how these helicases operate on their native substrates, namely protein-coated DNA.
Sectors Healthcare

Pharmaceuticals and Medical Biotechnology
 
Description An open lecture entitled Removing roadblocks to replication. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The audience for my talk was between 100-200 people, drawn from both the general public and members of the University. There was a general discussion with the audience after my presentation which lasted approximately 30 minutes.

This event generated reports in the local media.
Year(s) Of Engagement Activity 2014