Lecturer in Genome Dynamics, Faculty of Medicine & Health Sciences
Complete, accurate replication of the genome is essential for life. Eukaryotes, such as yeast and humans, have large genomes with millions of bases encoding the genetic information. To ensure complete replication of these genomes within the allowed time, the process of DNA replication starts at multiple sites along each chromosome, called replication origins. These replication origins are specialised DNA sequences that assemble the cellular machinery that then moves along the DNA reading and copying the genetic material. It is essential that the cell activates sufficient replication origins to ensure complete replication of the chromosomes. The importance of controlling replication origin activation is highlighted by the genome instability that may result from uncontrolled chromosome replication. Despite the importance of DNA replication origins we understand little about the DNA sequences that specify and control them. Failures in the processes of DNA replication lead to genetic instability and diseases such as cancer and congenital disorders.
Schematic representation of budding yeast chromosome VI. Circles represent replication origins, with larger circles representing more active origins (used in most cell cycles), yellow colouring indicates that the origin is activated early in S-phase and blue colouring indicates activation late in S-phase.
The differing requirement of replication origins for replication proteins:
Eukaryotic DNA replication initiates at multiple chromosomal sites called origins. S. cerevisiae origins, have differing requirements for replication proteins. By constructing chimaeric origins, I identified the sequences responsible for this effect: the core origin sequences and conflicting transcription. Genes flanking an origin modulate its requirement for replication proteins, with transcription towards an origin dramatically increasing the requirement. During this project, I developed methodologies for defining and characterising replication origin sequences.
Next: We are now extending this work to understand how the cell coordinates DNA transcription and replication.
Using comparative genomics to identify DNA replication origin sequences:
Several individual origins have been characterised, but no study had provided sufficient resolution to identify origin sequences genome-wide. S. cerevisiae origins have a modular structure with an essential sequence element called the ACS. I reasoned that a functional ACS would be more evolutionarily conserved than non-functional intergenic sequences. I identified phylogenetically conserved instances of the ACS and confirmed each as an origin using a novel, in vivo, high-throughput assay. This work pinpointed ~60% of S. cerevisiae origins allowing annotation of the S. cerevisiae genome (see www.yeastgenome.org).
Next: We are now using analogous techniques to identify replication origins in other organisms. In addition, this work provide a platform for understanding chromosome and genome replication, allowing us to investigate the properties of multiple replication origins to understand how they contribute to genome replication and stability.
Phylogenetic conservation of the essential origin sequence element at ARS305.
OriDB - a database of DNA replication origins:
I have developed an web-accessible database of S. cerevisiae origins. Published microarray datasets from US and Japanese laboratories and my own phylogenetic analysis underpin the database, which also incorporates older data from the literature. OriDB provides a valuable resource that has been welcomed by the DNA replication community and is also proving useful to others interested in chromosome research.
Next: We are expanding OriDB to include other chromosomal data and other model organisms.
OriDB, the Replication Origin Database.
Prof. Craig J. Benham (Mount Sinai School of Medicine, New York) - properties of DNA at replication origins.
Prof. Joel A. Huberman (Roswell Park Cancer Institute, Buffalo) - replication origins in fission yeast.
Prof. Ed Louis (University of Nottingham) - phylogenetic analysis of replication origins in budding yeasts.
Dr Alessandro de Moura (University of Aberdeen) - mathematical analysis of the dynamics of chromosome replication.
Prof. Katsuhiko Shirahige (Tokyo Institute of Technology) - genomic approaches to the study of chromosome replication.
Dr Tomoyuki Tanaka (University of Dundee) - single cell techniques for analysis of chromosome replication.