Rune Troelsgaard Pedersen:
The functional role of TopBP1 in DNA maintenance at mitosis

Date: 05-01-2015    Supervisor: Michael Lisby & Vibe Østergaard

When cells traverse mitosis, genome integrity of the emerging daughter cells is dependent on replication of the entire genome during the preceding S-phase and accurate chromosome segregation in mitosis. Replication stress may cause cells to enter mitosis with underreplicated loci, consisting of unreplicated DNA and DNA-repair intermediates. These loci can manifest themselves as breaks and gaps on metaphase chromosomes, which often coincide with specific chromosome loci termed common fragile sites (CFSs). Additionally, underreplicated loci function as physical links between sister chromatids, which can lead to anaphase bridges that impair accurate chromosome segregation.

The recent decade featured many advances in our understanding of how cells cope with underreplicated loci in mitosis. A major advance was the description of ultra-fine anaphase bridges (UFBs), a class of anaphase bridges that are devoid of histones and do not stain with DAPI. Some of these UFBs are induced by replication stress and interlink CFSs on segregating sister chromatids in anaphase. Another major advance was the discovery of active processing of underreplicated loci by structure-selective endonucleases MUS81 and GEN1. This active processing was found to be an underlying mechanism of CFS expression. A final advance was the description of how DNA damage, arising as a consequence of replication stress in S-phase, was shielded in 53BP1 nuclear bodies (NBs), preventing untimely DNA repair during the subsequent G1-phase.

We established Saccharomyces cerevisiae as a model organism to study anaphase bridges, and we identified Dpb11/TopBP1 as a novel UFB-associated protein in yeast and avian DT40 cells, respectively. TopBP1 localized to confined areas on replication-stress induced UFBs. Upon onset of mitosis we observed a burst in TopBP1 accumulation onto chromatin, which gradually dissociated from chromatin during progression through mitosis. A fraction of the TopBP1 foci persisted into G1, where they transformed into 53BP1 NBs. We established two new functional roles of TopBP1 at mitosis; (i) TopBP1 localizes at sites of active DNA synthesis at the G2/M transition where TopBP1 promotes DNA synthesis. (ii) TopBP1 colocalizes with the scaffold protein and structure-selective nuclease subunit SLX4, and is required for SLX4 recruitment to chromatin in mitosis. Depletion of TopBP1 at mitosis greatly induces formation of chromatin bridges in mitosis and 53BP1 NBs in the daughter cells, thus underlining the functional importance of TopBP1 at mitosis.

Collectively our data show that TopBP1 plays an important role at mitosis to ensure genome integrity. We propose that TopBP1 is recruited to underreplicated loci at mitosis to promote DNA synthesis and SLX4-mediated resolution of replication and repair intermediates. This insight substantially contributes to the understanding of how cells cope with underreplicated loci in mitosis.