Analysis of the role of CIZ1 in regulation of DNA replication

Tollitt, James and Copeland, Nikki (2021) Analysis of the role of CIZ1 in regulation of DNA replication. PhD thesis, Lancaster University.

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Abstract

Metazoan DNA replication is a finely controlled event. The driving force promoting cell division is increasing kinase levels as cells progress from G1 to Mitosis. Phosphorylation of cell cycle driving targets is driven though motifs surrounding phosphorylation sites in proteins. Dysregulation of these events results in an alteration of the DNA replication programme inducing DNA replication stress. This can result in an erosion of genome stability, driving cancer progression. Mutation in multiple oncogenes has the effect of altering CDK networks, and replication timing. CIZ1 is a DNA replication factor. It plays a role in co-ordinating the spatiotemporal localisation of Cyclin A and Cyclin E surrounding the G1/S transition. CIZ1’s activity is controlled by CDK mediated phosphorylations, when hypo-phosphorylated, CIZ1 promotes nuclear matrix localisation of Cyclin A. Evidence suggests that CIZ1 has both oncogenic and tumour suppressor functions. However, the mechanisms that link CIZ1 to cancer remain unclear. CIZ1 may act as a kinase sensor modifying how cells respond to CDK networks. Here, to determine the role of CIZ1 in cell cycle progression, and DNA replication two independent CIZ1 KO cell lines were produced. There were no detectable differences in cell cycle profiles using EdU and flow cytometry. However, analysis of cell cycle re-entry kinetics after release from quiescence showed that CIZ1 KO cells bypass restriction point earlier than parental cell lines. In addition, CIZ1 KO cells express cyclin A earlier than parental cell lines, consistent with the earlier timing of restriction point. Significantly, cell free DNA replication analysis of CIZ1 KO cell lines and parental cell lines revealed CIZ1 KO cells showed a defect in cyclin A chromatin loading that could be reversed by titration of CIZ1. This suggests that CIZ1 aids localisation of cyclin A to chromatin. Furthermore, CIZ1 KO cell lines require 2-fold higher cyclin A-CDK2 levels to initiate DNA replication in vitro, and this effect is reversed by addition of CIZ1 which reduces this level to the optimal level for the parental cells. In addition, analysis of the interplay between CIZ1 and cyclin A-CDK2 showed that the threshold CDK activity to promote DNA replication is modulated by the levels of CIZ1. Analysis of the DNA replication kinetics of CIZ1 KO cell lines by DNA combing identified a DNA replication stress phenotype, characterised by a change in DNA replication dynamics, including a reduced fork rate and increased origin firing. The DRS phenotype was reversed by CIZ1 addback (CIZ1AB). In addition, CIZ1 KO cells showed defects in recovery from DRS inducing agent hydroxyurea and this defect was reversed in CIZ1AB cell lines. Together this data indicates that CIZ1 is involved in regulating cellular responses to CDK activity, that CIZ1 KO induces altered replication timing and replication stress. These observations provide mechanistic insight into the role that CIZ1 plays in tumorigenesis.