Chromosomal Replication Group
Group Leaders: Professor Gareth H Williams and Dr Kai Stoeber
The initiation of DNA replication is a crucial decision point in cell proliferation. Chromosomal replication lies at the point of convergence of oncogenic signalling pathways and transduction pathways that trigger proliferation. Unlike these complex redundant pathways, there appears to be only a single mechanism of DNA replication initiation that is conserved in all eukaryotic species studied. This feature makes it a potentially attractive target for both diagnostic and therapeutic intervention.
Our investigation of the DNA replication licensing pathway in Homo sapiens is revealing fundamental new insights into the different growth control strategies adopted by self-renewing, stable and permanent tissues and their dysregulation in cancer. The clinical translational studies arising from these scientific investigations are strongly supporting our hypothesis that the replication licensing pathway (RLP), a final and critical step in growth control, is an attractive target for diagnostic exploitation and applicable to a wide range of cancers irrespective of genetic background and histological type. The research programme is divided into seven main projects, which provide the rationale for development of novel diagnostic and therapeutic strategies targeting the DNA replication licensing machinery.
Origin licensing during exit into quiescence and re-entry into the mitotic cell division cycle
The aim of this project is to investigate the mechanisms underlying loss of proliferative capacity as cells exit the cell division cycle into the resting quiescent state (G0), and to identify the causal molecular events that give rise to replication competence following re-entry into cycle (G0-S transition). We have previously investigated in detail the regulation of pre-replicative complex (pre-RC) assembly during the G0-S transition by combining a fibroblast quiescence model system with direct biochemical studies in our cell-free DNA replication assays. Our current focus is on the temporal sequence of molecular events involved in the transition from pre-RCs to pre-initiation complexes and the activation (firing) of licensed replication origins.
Origin licensing and loss of proliferative capacity during differention
Differentiation of metazoan cells implies cessation of DNA replication and cell proliferation. This growth strategy ensures that differentiated tissues have little to no regenerative capacity and cannot compensate for malfunction or loss of their structural units. Little is known about the molecular mechanisms by which activation of the differentiation programme is coupled to loss of proliferative capacity. The aim of this project is to extend our ongoing analysis of the linkage of the RLP with loss of proliferative capacity during withdrawal of cells from cycle into the terminally differentiated state. Elucidating how cells at certain stages of development or differentiation lose their ability to divide should indicate ways in which this state can be reversed for tissue regeneration or, alternatively, in the opposite direction to suppress malignancy.
Control of origin licensing in normal and neoplastic human tissues
The constituents of the DNA replication licensing machinery can be regarded as relay stations coupling growth regulatory pathways with the initiation of chromosomal replication. Regulation of the RLP is therefore a powerful mechanism for controlling cell proliferation in tissues of multi-cellular organisms. The aim of this component of the research programme is to extrapolate in vitro findings from our other projects to the role of RLFs in the growth regulation of human tissues and their dysregulation during tumourigenesis.
Investigation of a putative origin licensing checkpoint
Inhibition of the replication licensing machinery, an evolutionary conserved step in the control of cell proliferation, might at first sight be expected to have comparable effects on normal cells and cancer cells. However, inhibition of the licensing machinery in vitro produces unexpected and strikingly different effects in normal and transformed cells (Shreeram et al., 2002; Shreeram and Blow, 2003). In untransformed IMR-90 fibroblast cells, inhibition of origin licensing causes a cell cycle arrest in G1 phase. In contrast, inhibition of origin licensing in transformed Saos2 and U20S cells results in progression into late S and G2/M phase after which cells undergo apoptosis. These findings point towards an as yet unidentified ‘origin licensing checkpoint’ that monitors assembly of pre-RCs at a critical number of replication origin scattered along the chromosomes. Exploiting a novel delivery system for a non-degradable derivate of the endogenous origin licensing inhibitor geminin, we are investigating the origin licensing checkpoint in normal and transformed human cell lines. These studies will determine whether the origin licensing machinery is a promising novel therapeutic anti-cancer target.
Cdc7 small molecule inhibitor screening programme
Cdc7 activity is regulated by ASK (the human analogue of yeast Dbf4) and plays an essential role in assembly of the pre-RC by phosphorylating MCM proteins. In the absence of Cdc7 activity the latter stages of pre-RC assembly would not be possible, and the cell would not proceed through the G1-S transition to begin DNA synthesis. Consequently inhibition of Cdc7 activity represents an attractive target for an anti-proliferative agent. In collaboration with Cancer Research Technology Limited (CRT) we have developed a primary screening assay for Cdc7 based on measuring the ability of small molecules to inhibit phosphorylation of a generic serine/threonine kinase substrate. The assay itself is a competition assay between potential inhibitors and a known, broad-spectrum kinase inhibitor (acting at the ATP binding site) such as staurosporine, the latter being coupled to a reporter system such as a ß-galactosidase fragment. CRT has a large screening collection of diverse molecules, which are being employed in the primary assay to search for hits. However the throughput of the primary assay, the availability of detailed structural information for related proteins, and the extensive medicinal chemistry literature relating to known kinase inhibitors suggests that a more directed screening strategy would be successful, and may offer a quicker, more economical route for hit identification. We have already built and validated a homology model for Cdc7, based on the structural information for other protein kinases, and we are using this model to select templates that we anticipate can provide Cdc7 inhibitory activity.
Structural biology of the preRC complex assembly and function
We have previously successfully employed a combination of cellular and structural molecular biology based approaches to advance our understanding of the molecular architecture and function of licensing factors that form the DNA replication initiation machinery. Electron microscopy (EM) combined with single particle analysis have produced the first 3D structure of full-length human geminin. In collaboration with the School of Crystallography at Birkbeck College and the Institute of Structural Molecular Biology (London) we continue to use this approach to address the structure-function relationship of other components of the DNA replication initiation machinery responsible for the transition from pre-RCs to pre-ICs and origin firing.
Regulation of mitosis by phosphorylation
We are part of the EU 6th Framework Programme (FP6) Integrated Project ‘MitoCheck’ which is a combined functional genomics, proteomics and chemical biology approach to study the regulation of mitosis by phosphorylation (see also www.mitocheck.org).
Cell division (or “mitosis”) is one of the fundamental processes of life. Mistakes during cell division contribute to cancer and, if they occur during reproduction, are the leading cause of infertility and mental retardation. However, it is still poorly understood how cells coordinate many disparate but inter-locking processes during mitosis. One of the well established facts is that protein kinases – a certain type of enzymes – play a major role in cell cycle regulation. It has long been suspected that by modifying different substrates, protein kinases help to orchestrate many mitotic processes, but what their key substrates are remains largely unknown. MitoCheck has been established to systematically search for proteins crucial for mitosis, to determine how these proteins are modified by phosphorylation – which is what kinases do – and to asses whether mitotic kinases have any diagnostic or prognostic potential in cancer treatment.
MitoCheck is the largest integrated research project on cell cycle control within the 6th Framework Programme (FP6) of the European Union. Leading scientists from eleven top research institutes, universities and industry in Austria, Germany, UK, Italy and France (such as EMBL, IMP, Sanger Institute, CBG and others) with a wide range of expertise in molecular and cell biology, biochemistry, modern microscopy techniques, proteomics, bio-informatics and clinical pathology join forces to take on the challenge of unravelling the mystery of cell division.
Current and Past Group Members
Williams GH & Stoeber K (2012) The Cell Cycle and Cancer. J Pathol 226:353-64. Review Pubmed
Tudzarova S, Colombo SL, Stoeber K, Carcamo S, Williams GH, Moncada S (2011) Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle. Proc Natl Acad Sci USA 108:5278-83. Pubmed
Tudzarova S, Trotter MW, Wollenschlaeger A, Mulvey C, Godovac-Zimmermann J, Williams GH, Stoeber K (2010) Molecular architecture of the DNA replication origin activation checkpoint. EMBO J 29:3381-94. Pubmed
Rodriguez-Acebes S, Proctor I, Loddo M, Wollenschlaeger A, Rashid M, Falzon M, Williams GH (2010) Targeting DNA replication before it starts: Cdc7 as a therapeutic target in p53-mutant breast cancers. Am J Pathol 177:2034-2045. Pubmed
Roberts S, Kingsbury SR, Stoeber K, Knight GL Gallimore PH & Williams GH (2008) Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells. J Virol 82:9056-9064. Pubmed
Okorokov AL, Waugh A, Hodgkinson J, Murthy A, Hong HK, Leo E, Sherman MB, Stoeber K, Orlova EV, Williams GH (2007) Hexameric ring structure of human MCM10 DNA replication factor. EMBO Rep 8:925-930 Pubmed
Stoeber K, Swinn R, Prevost AT, de Clive-Lowe P, Halsall I, Dilworth SM, Marr J, Turner WH, Bullock N, Doble A, Hales CN, Williams GH (2002) Diagnosis of genito-urinary tract cancer by detection of minichromosome maintenance 5 protein in urine sediments. J Natl Cancer Inst 94:1071-1079 Pubmed
Williams GH, Romanowski P, Morris L, Madine M, Mills AD, Stoeber K, Marr J, Laskey RA, Coleman N (1998) Improved cervical smear assessment using antibodies against proteins that regulate DNA replication. Proc Natl Acad Sci USA 95:14932-14937 Pubmed