Origin usage in a human cell-free system

Lines connect the coordinator with its partners. CORDIS does not always give exact coordinates for projects before 2014. These points are placed at city or country level.

Project objective

Research objectives and content DNA replication is a process of dramatic interest for its relevance in the transmission of genetic information from one cell generation to the next. Unfortunately, this process in human cells is still poorly known. However, very recently, the first human system that supports replication in vitro has been developed in R.A.Laskey laboratory. In this cell-free system nuclei from G1 phase HeLa cells initiate semiconservative DNA replication in the presence of cytosolic and nuclear factors from S-phase cells. Exploiting the skills and the knowledge of the laboratory that developed the cell-free system, I propose to utilize the same to understand the nature of DNA replication initiation in human cells. Basically, I will utilize three approaches: - origin usage studies including replication timing analysis and mapping of origins at the DNA level - replication assays using different templates to establish an in vitro system able to replicate not only DNA presented as Gl nuclei, but also DNA provided as another source - biochemical approaches including fractionation, addition and selective immunoprecipitation of target proteins. These approaches will allow me to evaluate the importance of DNA sequences versus chromosomal and nuclear structures in selection of replication origins and to explain the roles of initiation proteins. Training content (objective, benefit and expected impact) The newly developed human cell-free system will be crucially important in the dissection of the S-phase cellular controls as well as the molecular mechanisms underlying DNA replication. Moreover, the same will be an important approach to study other relevant cellular processes such as DNA recombination and repair. Links with industry / industrial relevance (22) Possible links with the industry could be the use of this assay in investigating the effects of anti-tumor drugs and also the effects of different stimuli, such as X-ray irradiations, on the control mechanisms of DNA replication and, as a consequence, on

Original text from CORDIS.

Participants

• University of Cambridge · CambridgeCoordinatorUnited Kingdom
• Not availableCity levelItaly

Links

• View on CORDIS