Investigations into the spatial distribution of γH2AX around a DNA double-strand break and the analysis of double-strand break mobility

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation by the protein kinase ATM. H2AX is unevenly distributed throughout chromatin and is rapidly phosphorylated to form γH2AX up to 2 megabases either side of DSBs. Studies in yeast systems have shown that while γH2A can spread in cis surrounding the break site, it can also spread in trans onto unbroken chromosomes located in close spatial proximity. Although the majority of data in the current literature presents the well characterised in cis spread of γH2AX, there are strong indications that it can also occur in trans in mammalian systems; analogous to the findings shown in yeast. This thesis lays out the steps taken to develop a novel system to address the spatial distribution of γH2AX around a nascent DSB. Since the first published live imaging experiments of the dynamics of chromatin by in vivo single particle tracking there has been extensive investigation into the regulation and biological function of movement of damaged DNA. In yeast, a relative consensus exists that DSB induction increases the movement of a DSB. In contrast to yeast however, data published of DSB movement in higher eukaryotes has been controversial, caused by conflicting results. Here, I developed a cell-based system, and utilised timelapse live cell imaging to show that a chromosomal locus containing a single endonuclease-induced DSB shows confined movement in comparison to an undamaged locus. Furthermore, this confined movement of a damaged locus is compounded by treatment with an ATM kinase inhibitor but not a DNA-PKcs kinase inhibitor, suggesting that the kinase activity of ATM and not the kinase activity of DNA-PKcs plays a significant role in the dynamics of DSBs

Another string to the polo bow: a new mitotic role of PLK1 in centromere protection

Polo-like kinase 1 (PLK1) plays a fundamental role in the spatiotemporal control of mitosis. Cells lacking PLK1 activity exhibit characteristic chromosome misalignment due to defects in microtubule-kinetochore organization and attachment. In our recently published paper, we uncover a new role for PLK1 in the preservation and maintenance of centromere integrity

DNA replication is highly resilient and persistent under the challenge of mild replication stress

Mitotic DNA synthesis (MiDAS) has been proposed to restart DNA synthesis during mitosis because of replication fork stalling in late interphase caused by mild replication stress (RS). Contrary to this proposal, we find that cells exposed to mild RS in fact maintain continued DNA replication throughout G2 and during G2-M transition in RAD51- and RAD52-dependent manners. Persistent DNA synthesis is necessary to resolve replication intermediates accumulated in G2 and disengage an ATR-imposed block to mitotic entry. Because of its continual nature, DNA synthesis at very late replication sites can overlap with chromosome condensation, generating the phenomenon of mitotic DNA synthesis. Unexpectedly, we find that the commonly used CDK1 inhibitor RO3306 interferes with replication to preclude detection of G2 DNA synthesis, leading to the impression of a mitosis-driven response. Our study reveals the importance of persistent DNA replication and checkpoint control to lessen the risk for severe genome under-replication under mild RS

<b>Research data for Centromere protection requires strict mitotic inactivation of the Bloom syndrome helicase complex</b>

This record contains the figures to support the article. Article abstract The BTRR (BLM/TOP3A/RMI1/RMI2) complex resolves various DNA replication and recombination intermediates to suppress genome instability. Alongside PICH, they target mitotic DNA intertwinements, known as ultrafine DNA bridges, facilitating chromosome segregation. Both BLM and PICH undergo transient mitotic hyper-phosphorylation, but the biological significance of this remains elusive. Here, we uncover that during early mitosis, multiple protein kinases act together to strictly constrain the BTRR complex for the protection of centromeres. Mechanistically, CDK1 destabilises the complex and suppresses its association with PICH at the chromatin underneath kinetochores. Inactivating the BLM and TOP3A interaction compromises the UFB-binding complex mitotic functions and can prevent centromere destruction. We further unravel how different clusters of mitotic phosphorylation on BLM affect its interaction with the TOP3A/RMI1/RMI2 subcomplex and illegitimate centromere unwinding. Furthermore, we identify specific phosphorylation sites targeted by the MPS1-PLK1 axis functioning to prevent BLM hyper-activation at centromeres. Notably, unleashing such activity after sister-chromatid cohesion loss facilitates separation of entangled chromosomes. Together, our study defines a centromere protection pathway in human mitotic cells, heavily reliant on a tight spatiotemporal control of the BTRR complex.</p