Torsten Krude - Overview
Our research focuses on the molecular control mechanisms of chromosome replication in human proliferating somatic cells, using biochemical and cell biological methods.
A key event in the regulation of the cell division cycle is the initiation of genomic DNA replication in the cell nucleus at the G1 to S phase transition, where the cell is committed to a full duplication cycle. Early cell fusion studies had shown that G1, but not G2 phase nuclei are triggered to prematurely initiate DNA replication when fused to S phase cells, establishing that nuclei are competent to initiate DNA replication in G1 phase and that soluble initiation factors are present in S phase. We have established and are now exploiting an experimental in vitro system derived from human tissue culture cells that initiates DNA replication in isolated cell nuclei under the control of the cell division cycle.
Initiation of DNA replication in our in vitro system can be triggered in nuclei isolated from late G1 phase cells, upon incubation in cytosolic extract from proliferating cells. Initiation depends upon the G1/S phase-specific protein kinases cyclin A/Cdk2 and cyclin E/Cdk2 and upon additional soluble factors present in the extract. Our current main focus in the lab is to identify and purify these additional factors by biochemical fractionation of the cytosolic extract. This approach is set up to isolate soluble factors that control the establishment of DNA replication forks within the context of initiation-competent late G1 phase chromatin in the functional constraints of the cell nucleus.
DNA replication starts at many thousand sites, or replication origins, in the human genome. We have recently found in a collaborative work that initiation of DNA replication in our cell-free system is spatially and temporally controlled. Initiation in vitro occurs only at those same specific sites that are used in vivo at the beginning of S phase. We are currently investigating further the constraints of this site-specificity of initiation and have begun to isolate and characterise DNA sequences from the human genome, which are used as DNA replication start sites.
An additional point of our research is the assembly of newly replicated DNA into chromatin. Efficient chromatin assembly is essential for the cell to pack the genomic DNA into the confines of the cell nucleus and to allow regulation of gene expression and replication to take place. New nucleosomes are assembled by chromatin assembly factors from newly synthesised histone proteins during S phase of the cell division cycle. We are currently investigating how the cell cycle control machinery regulates nucleosome assembly during DNA replication, again by using cell-free systems derived from human cells. We were able to demonstrate that nucleosome assembly mediated during DNA replication by a key factor, the histone chaperone CAF-1, requires reversible phosphorylation by cyclin-dependent protein kinases (cyclinA-Cdk2 and/or cyclinE-Cdk2) and dephosphorylation by protein phosphatase 1 (PP1). Therefore, the same protein kinases complexes that are required for initiating genomic DNA replication at the G1 to S phase transition are also and required for the assembly of newly replicated DNA into chromatin. We are currently aiming at dissecting this regulatory pathway further.
