The regulation of chromosome replication by DBF4-Dependent Kinase

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Defects in DNA replication during the process of cell division can lead to long-term problems, including loss of genome integrity, irreparable DNA damage, cell cycle exit and senescence. The project will address how genome integrity is ensured during cell division, in particular how chromosomal DNA replication is controlled and coordinated with other cell cycle events. An important regulator of DNA replication is the DBF4-Dependent Kinase (DDK) which controls replication initiation and coordinates it with other cell division events such as sister chromatid cohesion. In the last few years our lab has made some important advances in understanding the function of DDK and how it regulates DNA replication (see Volpi et al, Open Biol. 11: 210121; Alver et al, Cell Reports 8: 2508-2520; and Poh et al, Open Biology 4: 130138). The project will build on this work to understand in more detail the way that DDK regulates the activity of key replication initiation proteins and coordinates this with chromosome cohesion. The research will primarily use Xenopus (frog) egg extracts that support all of the key nuclear events of the cell division cycle in vitro, which provide an unparalleled system to study genome integrity at a biochemical level. Results obtained from the Xenopus system have been shown to be widely applicable to other vertebrate cell types. Published results show that in the Xenopus system, DDK is required for both replication initiation and the establishment of sister chromatid cohesion, though the precise biochemical mechanisms are not well understood. We have an extensive range of reagents to study DDKs and their potential substrates, including specific antibodies for immunoprecipitation and immunodepletion as well as highly specific DDK small molecule inhibitors. We will use these reagents in combination with the world-leading mass spectrometry facility in Dundee (http://proteomics.lifesci.dundee.ac.uk) to identify novel DDK substrates that are involved in regulating DNA replication and the establishment of sister chromatid cohesion. We will use this information to build hypotheses about how these processes are coordinated and then test them in the Xenopus cell-free system. We will aim to take the key conclusions obtained from the in vitro work and examine whether they are conserved in human tissue cells.