Organisation of the initiation of DNA replication

• Main Author: Wilson, Rosemary Helen Clare
• Other Authors: Coverley, Dawn
• Published: University of York 2013
• Subjects: 572.8
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589210

Multiple lines of evidence show that DNA replication and the proteins involved with its preparation reside at the nuclear matrix (NM). Some of these, such as cyclin E, are recruited to the NM during differentiation, implying that NM attachment may help to fix cell-type specific replication programmes, and potentially therefore restrict plasticity. However, our understanding of the interplay between the NM, the cell cycle and cell type is still limited. The aim of this PhD is to develop our understanding of the NM in relation to the preparation for DNA replication. The function of the replication-promoting and cancer-associated NM protein CIZ1 was investigated using cells derived from a CIZ1-/- mouse. Dysregulated cyclin and CDKi expression and immobilisation suggest that CIZ1 has a widespread role as a cell cycle regulator with an exchange function that is consistent with previous analysis of its interaction with cyclin E and A. More importantly, in the absence of CIZ1, the DNA damage response is activated as cells receive growth inhibition signals, leading to the formation of foci outgrowths (and lymphoid malignancies in the animal). Together with published data, this suggests a tumour suppressor role for CIZ1. This also implies that different forms of CIZ1 have tumour suppressor or promoting roles and that the balance between these may be critically important for proper integration of growth control signals. CIZ1 has also previously been implicated in chromatin loop organisation during G1-phase. My analysis of NM attached chromatin loops during cell cycle re-entry from quiescence shows that average chromatin loop size is smaller in quiescence but recovers in size in a very early, growth factor-independent step during G1. Moreover, chromatin loops were also found to become larger and more unstable following controlled transformation, correlating with changes in CIZ1 expression. This suggests weaker attachment of DNA in cancer cells and mirrors the lack of attachment of cyclin E and CIZ1. These data support the hypothesis that loss of NM attachment is a feature of cancer cells and were collected using novel methods developed and validated during this project that allow rapid, non-subjective computer measurement of chromatin loops.