|Title:||Human GINS, a conserved DNA replication factor and candidate cancer marker|
|Supervisors:||Mills, Anthony D.|
Laskey, Ronald A.
|Abstract:||The GINS complex (a heterotetramer of Sld5, Psf1, Psf2 and Psf3) is a highly conserved DNA replication factor required for the initiation and elongation of DNA replication. GINS is believed to associate with Cdc45 and MCM proteins on replicating DNA. The interaction between GINS and MCM is also conserved in archaea. In my thesis, I explore the subcellular localisation of the GINS complex in relation to the MCM proteins and sites of DNA replication by high-resolution confocal microscopy. For these studies, I generated and carefully validated purified rabbit polyclonal and mouse monoclonal antibodies; these show a specific staining pattern by immunohistochemistry, immunoblotting and immunofluorescence. At high-resolution, all GINS antibodies produced a focal nuclear pattern, similar to that seen for the MCMs. However, confusingly, colocalisation between GINS and MCMs and between the GINS subunits themselves is poor. Investigations are continuing to understand this conundrum. Given the value of MCM proteins as specific and sensitive markers for cancer screening, I investigated whether GINS subunits also have potential diagnostic value. Sld5 and Psf3 expression is restricted to the proliferative compartment in normal tissue, but is found in the majority of cells in a wide range of dysplastic and malignant tissues, including cervix, colon and bladder. In vitro studies of tissue culture cells and cell lysates incubated in urine suggest that Sld5 protein is more stable than Mcm2 in harsh extracellular environments. In an ongoing pilot clinical study of Sld5 protein as a potential biomarker, Sld5 is readily and specifically detectable in the cellular fraction of the samples from prostate and bladder cancer patients. Work is ongoing to evaluate Sld5 protein levels in the supernatant portion of those same urine samples as an easy-to-screen diagnostic/prognostic marker for male urogenital cancers. Owing to their stability, GINS proteins hold promise as independent or complementary markers to the MCM proteins for cancer screening in harsh extracellular environments such as urine.|
|Appears in Collections:||Theses - MRC Cancer Cell Unit|
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