How Do Cells Control the Temporal Programme of DNA Replication?

All cells contain a complete copy of the organism?s DNA packaged into units called chromosomes. This DNA is often called the ?genome?, and contains the information which is the genetic blueprint of life. All new cells need a copy of the genomic DNA, and so the DNA must be completely and accurately copied for successful cell multiplication. Interruptions and errors during replication of the DNA can lead to ?genome-instability? diseases such as cancer. DNA replication initiates at multiple sites along each chromosome, called replication origins. These origins are specialized chromosome locations at which DNA-copying machines can be assembled, after which these machines move along the chromosome replicating the DNA as they proceed. We would like to understand how the DNA replication process is controlled, and how defects in this process can lead to disease. We study DNA replication in the budding yeast S. cerevisiae, which is an excellent ?model organism? because its gene structure is simple, and its replication origins are particularly well understood. Intriguingly, DNA replication takes place according to a predetermined programme, with some replication origins initiating much earlier than others. Some cancer cells show defects in the mechanisms that control this temporal programme. We do not understand how cells distinguish early and late origins, although chromosome context appears to be important for correct initiation time. For example, origins that lie close to chromosome ends tend to initiate replication late. We discovered a cellular component (called ?Ku?) that is important for making origins close to chromosome ends late-initiating. Ku binds the very tips of chromosomes but affects the initiation time of origins a surprising distance away. The aim of this research is to understand how Ku affects replication origin initiation time. We will test whether Ku brings about changes in how the DNA surrounding origins is packaged; we might expect to find that tight packaging of the DNA close to an origin causes it to initiate replication late. We will also test whether the length of the chromosome tips affects the replication programme, and examine how events at the chromosome end are propagated along chromosomes to replication origins. This type of ?chromosome context? effect has emerged in recent years as crucially important for the biological function of DNA. As well as improving our understanding of mechanisms that cause cancer, the result of our research will be useful to scientists developing strategies for gene therapy.

The entire complement of genomic DNA must be accurately replicated at the correct time in the cell cycle to enable successful cell division. Errors or inaccuracies in DNA replication are a major cause of genome instability diseases such as cancer. We aim to understand how the process of DNA replication is controlled. Replication initiates from multiple sites on eukaryotic chromosomes called replication origins. The various replication origins are activated according to a specific temporal programme, with some reproducibly initiating replication early and others only much later. DNA replication time is related to transcriptional status, and cells employ checkpoint controls to ensure that DNA is replicated in the correct order. Derailment of such checkpoint pathways is commonly observed in cancer. We are investigating the molecular mechanisms by which replication origins are directed to initiate early or late, using yeast as a model system because it has the best characterised eukaryotic replication origins. Origin initiation time is strongly influenced by chromosome context, with origins in certain contexts (e.g. close to one of the telomere structures that terminates chromosomes) tending to initiate late. We discovered that a telomere-bound protein complex called Ku is crucial for the correct, late initiation time of origins up to 40 kilobases from the telomere. The aim of this work is to understand how Ku, which is localised at telomeres, affects replication origins over such a long distance. Ku is necessary for maintaining the terminal sequence repeats of yeast chromosomes at their correct length. First, by manipulating telomere length in various strains we will test whether Ku affects telomere-proximal replication origins through its role in telomere length maintenance. Modifications to chromatin structure have been suggested to affect origin initiation time, and so second, we will use chromatin immunoprecipitation and nucleosome mapping techniques to examine chromatin structure at replication origins, to test whether telomeric Ku mediates changes in origin chromatin configuration. Third, we will investigate mechanisms by which molecular events at telomeres are transmitted to origins tens of kilobases distant, by examining the effects of mutations implicated in long-range chromosome organisation. This work will inform our understanding of the cell cycle by illuminating the controls over DNA replication, a well as revealing how chromosome context and higher-order chromatin structure exert long-range effects on genome function.