Single molecule analysis of genome replication

All cells contain a complete copy of the organism's DNA, the genetic blueprint of life, packaged into discrete units called chromosomes. Since new cells need a copy of the genetic material, the chromosomes must be completely and accurately replicated before the cell can divide. Our research aims to determine how cells ensure that the replication of each chromosome is completed before cell division. Our experimental data have revealed the average pattern of chromosome replication in populations of millions of cells - that is the regions that are replicated first, second and so on. However, in preliminary data we have discovered that individual cells do not replicate their chromosomes with this 'average' pattern; the pattern of DNA replication in single cells is hidden by the population average. Consequently rare problems during DNA replication, including delays in completing replication, cannot currently be readily detected. Therefore, we are developing novel methods to directly measure DNA replication in single molecules. In one approach, we are using a transformative single-molecule sequencing technology to detect the pattern of DNA replication in thousands of long molecules. In addition, we will make use of recent advances in imaging technologies to directly visualise DNA replication in whole chromosomes. These complementary approaches will, for the first time, reveal the pattern of DNA replication on individual chromosomes. This is important because a single DNA replication error on one chromosome in a single cell division can give rise to genomic disorders, including cancer.

Complete, accurate genome replication is essential for life. Our long-term goal is to determine how cells faithfully complete genome replication. Errors in DNA replication occur on single molecules in individual cells; however these errors are hidden from view in genomic approaches that look at data from populations of several million cells. Currently, there are no genomic methods to study DNA replication in single cells or molecules. We will close this gap by pioneering two independent single-molecule techniques to discover how the genomes of individual cells are replicated. First, we will exploit recent dramatic advances in single-molecule DNA sequencing to directly detect base analogues that were incorporated during a pulse-chase experiment. This builds upon our demonstration that the MinION nanopore sequencer can clearly distinguish between natural bases (such as thymidine) and a base analogue (such as bromodeoxyuridine, BrdU) on ultra-long DNA sequence reads (>100 kb). Second, we will make use of a high-resolution DNA visualisation platform (called Irys) to detect the pattern of DNA replication in chromosome-length molecules. This will allow us to determine the location of active replication origins and the location of replication termination sites, in thousands on individual molecules. Together these experiments will provide the first high-resolution, whole-genome view of chromosome replication in single molecules. Our work with both wild-type and perturbed cells will allow us to discover mechanisms that contribute to stable genome inheritance.

Who will benefit from this research?

The proposed work has long-term healthcare implications that will be of potential benefit to a wide range of patient groups, in particular cancer sufferers. Disruption of the regulation of DNA replication contributes to genome instability by leading to chromosome breaks, translocations and aneuploidy. Genomic regions with few active replication origins are hotspots for rearrangements in cancer. Outcomes from the proposed research will provide novel mechanistic insights to genome replication and the human diseases associated with its deregulation, including cancer and developmental disorders. Thus, the biomedical implications of this work fit within the BBSRC's Strategic Research Priority "Bioscience for health".

How will they benefit from this research?

This proposal aims to investigate chromosome replication in single molecules and determine the degree of cell-to-cell variability. We will work in the genetically tractable model system Saccharomyces cerevisiae. We anticipate that our results will be informative about genome replication in other eukaryotes, because the key proteins involved in binding and activating replication origins are conserved between yeast and humans. Furthermore, differences in replication time (and the features that they correlate with) are also found from yeast through to humans. Therefore, the results of this project could help determine why some genomic regions have a low abundance of active replication origins and this could be an essential step towards improved therapeutic intervention.

What will be done to ensure that they have the opportunity to benefit from this research?

In addition to traditional routes of publication, the outcomes from this project will be communicated through our web pages, the replication origin database (OriDB), the University of Oxford's Communications and Marketing Unit, Oxford's Café Scientifique, and the BBSRC media office. Potential future health benefits will be exploited via colleagues within the Medical Sciences Division.

Professional development for staff working on the project

The project offers many opportunities for the postdoctoral researchers to acquire additional skills. The collaborative nature of the research will expose both individuals to molecular biology, genomics, bioinformatics and mathematical modelling. The PDRAs will receive training and hands-on experience of advanced bioinformatics approaches for analysis of novel deep sequencing technologies. The scientific communication skills of the PDRAs will be fostered by presenting our research to academic audiences and the general public (e.g. Oxford's Café Scientifique or local schools). Appropriate training for both audiences will be provided by the University of Oxford Science Outreach Programme, at a Genetics Society Workshop on 'Communicating Your Science' and at a EMBO Laboratory Management Course for Postdocs.