What regulates replication origin activation?

All cells contain a complete copy of the organism's DNA, the genetic blue print 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. 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. I hope that a better understanding of the basic biology that ensures genetic integrity will give new insights that will allow improved diagnosis and treatment of these diseases. In addition to DNA replication, the genetic material is also read and then translated to make proteins. The initial step in this process is called transcription. I have recently found that transcription is detrimental to replication origin function and may therefore play a key role in determining which DNA sequences can function as origins. This project aims to understand how the cell coordinates the two key processes that read the genetic information, DNA replication and DNA transcription, to ensure genomic stability. I will work with budding and fission yeasts, because their genomes are well understood and easily modified to ask experimental questions, and importantly the controls over DNA replication are similar to those in human cells. Furthermore, I have already precisely identified the location of more than half of the budding yeast replication origins providing a large dataset to help me understand the properties of replication origins. By collaborating with leading fission yeast laboratories I will identify the location of replication origins in this species. This will allow, for the first time, genome-wide comparisons of replication origin characteristics between two organisms to determine which properties are shared and therefore likely to be of functional importance. I will go on to look directly at how replication is affected by transcription and what molecular mechanisms are used by the cell to protect replication, and specifically replication origins, from transcription. These experiments will not only allow me to understand how the cell coordinates replication and transcription, but will also give an understanding of what determines replication origin behaviour at the molecular level. Using these results, I will build a computer-based model of the processes of chromosome replication and test the model by comparing the computer predictions with experimental results. Differences between prediction and observation will highlight the limitations in our understanding of DNA replication, indicating important directions for further experiments. This work will uncover how DNA replication origins are specified and how their behaviour is regulated. By understanding, at the molecular level, the processes that control replication origins throughout the genome I will be able to model how whole chromosomes are replicated. This model will allow me to predict weaknesses in the chromosome replication process that may underlie genetic diseases such as cancer.

Complete, accurate genome replication is crucial for successful cell division and continuation of life. DNA replication is controlled by regulating activation of replication origins to give bi-directional replication forks that must stably progress to replicate the DNA. Transcription is detrimental to both origin activation and fork progression in all studied systems, including E. coli, yeasts and metazoans. Recent work suggests that transcription may be important in defining replication origin sites in both yeast and metazoans. Therefore, understanding how the cell coordinates DNA replication and transcription is crucial for understanding cell division and genome stability. The interplay between replication and transcription is subject to both predefined and regulatable controls. For example, chromosome structure defines the relative location of transcription units and replication origins, whereas the cell can regulate when and where to activate origins and express transcription units. I aim to understand the relationship between transcription and replication using the following approaches. (1) I will determine the predefined aspects of the replication system by identifying the location of origins in fission yeast and by comparing their characteristics with budding yeast origins. Then, initially in budding yeast, I will quantify the regulatable variables that determine origin usage by: (2) investigating the role of transcription in regulating origin activity and (3) analysing origin activation time and efficiency genome-wide to understand their relationship. (4) Comparison of these data with computer simulation of chromosome replication will test the completeness of understanding and highlight discrepancies for further investigation. This will require investigation of replication dynamics in single cells to understand the stochastic properties of DNA replication.