Characterization of the regulation of DNA joint molecule resolution during cell cycle and in response to replication fork stalling

The DNA is the central cellular information carrier, but its stability is constantly challenged by DNA-damaging incidents. As DNA lesions may elicit genomic instability – in mammalian cells the cause of cancer – DNA repair processes are indispensable for cellular integrity. DNA lesions during S-phase are a particular detriment as they interfere with replication fork progression and faithful chromosome duplication. Fork stalling at DNA damage sites is a common perturbation during replication, but may be bypassed by recombination-based mechanisms. These pathways involve the undamaged sister chromatid as a recombination template and as such formation of intermediate DNA structures, so-called DNA joint molecules (JMs) between both chromatids. Such covalent DNA linkages need to be disentangled before chromatid separation in anaphase to avoid chromosome breakage. Two principle mechanisms have been described to disentangle DNA JMs: dissolution, comprising collaborative helicase-topoisomerase activity, and resolution, comprising cleavage by endonucleases such as Mus81-Mms4 or Yen1. Previous research has revealed that JM resolvase activity by Mus81-Mms4 is under stringent cell cycle control, and up-regulated specifically in mitosis upon CDK- and Cdc5-dependent phosphorylation. Yet, we are only beginning to unravel the molecular mechanism of this temporal regulation. In this study, we identify distinct means how cell cycle signals can be integrated into the activity of the JM resolvase Mus81-Mms4 using Saccharomyces cerevisiae as a model organism. First, we discovered a third cell cycle kinase, which is crucial for Mus81 nuclease activation in mitosis: Cdc7-Dbf4 (DDK, Dbf4-dependent kinase) targets Mus81-Mms4 together with Cdc5. Both kinases bind and phosphorylate Mus81-Mm4 inter-dependently in order to promote full Mus81 activation. A second layer of the temporal control of Mus81 is mediated by scaffold proteins. Cell cycle-dependent phosphorylation induces the formation of a multi-protein complex comprising the scaffold proteins Dpb11, Slx4, and Rtt107. Already in S-phase during the response to replication fork stalling, those proteins interact with each other upon evolutionary conserved CDK phosphorylation of Slx4 that mediates binding to Dpb11. This S-phase complex has been implicated in the regulation of the DNA damage checkpoint after replication fork stalling and may have a potential DNA repair function. In M-phase, the scaffold complex associates with Mus81-Mms4 dependent on cell cycle kinase activity. We could show that the scaffold protein Rtt107 recruits the DDK-Cdc5 kinase complex to Mus81-Mms4 via a direct interaction between Rtt107 and Cdc7, enabling Mus81-Mms4 hyper-phosphorylation and Mus81 activation. Future research will need to identify additional regulation factors that may influence substrate specificity or targeting. Taken together, my PhD work described several regulatory mechanisms of mitotic DNA JM resolution by Mus81-Mms4 that involve the cell cycle kinases CDK, Cdc5 and DDK as well as the scaffold proteins Dpb11, Slx4, and Rtt107. These control mechanisms are highly inter-connected as association of the scaffold proteins depends on cell cycle kinase activity, but in turn stable multi-protein complex formation is required for efficient interaction with the DDK-Cdc5 kinases, full phosphorylation of Mus81-Mms4 and timely JM resolution.