Role of DDK kinase in DNA double-strand break repair and insights into the DDK-Cdc5/PLK1 kinase complex

Galanti, Lorenzo (2022): Role of DDK kinase in DNA double-strand break repair and insights into the DDK-Cdc5/PLK1 kinase complex. Dissertation, LMU München: Fakultät für Biologie

Abstract

The eukaryotic cell cycle consists of an ordered sequence of tightly regulated events to restrict specific activities within specific cycle phases. Key regulators are cell cycle kinases. Budding yeast harbor three essential cell cycle kinases conserved in humans: Dbf4-dependent kinase Cdc7 (DDK), Cyclin-dependent kinase (CDK) and the single yeast Polo-like kinase, Cdc5 (PLK1 in human). DNA double-strand breaks (DSBs) are a severe form of DNA damage. Two main pathways evolved for the repair of such toxic lesions are homologous recombination (HR) and non-homologous end joining (NHEJ). HR often uses the homologous sequence of the sister chromatid as template for error-free DSB repair. Therefore, HR is upregulated in S, G2 and M phase when a sister chromatid is present, while NHEJ is the preferred repair pathway in G1. The crucial switch from repair via NHEJ to HR is considered to be the processing of the broken ends during DNA end resection, which primes for repair by HR and inhibits repair by NHEJ. Part of this regulation comes from CDK phosphorylation of Sae2-MRX (CtIP-MRN in human), which initiates DNA end resection. However, it is increasingly clear that additional cell cycle regulated mechanism might be involved in the regulation of DNA end resection initiation. To identify novel functions of DDK, we performed phosphoproteomic experiments and discovered that DDK phosphorylates several proteins involved in DSB repair via HR, among which also factors involved in DNA end resection. We therefore followed a first line of research focused on the possible role of DDK in regulating DNA end resection. We showed that DDK is required for resection and HR, unveiling a previously unknown role of DDK in the cell cycle regulation of DSB repair. Mechanistically, we focused on phosphorylation of Sae2. We showed DDK-dependent phosphorylation of Sae2 in vivo and in vitro, and found that via phosphorylation of Sae2, DDK can stimulate the nucleolytic activity of the Sae2-MRX complex. Given the importance of DDK as regulator of resection, we tested if we could bypass the cell cycle regulation of DNA end resection and HR by forcing DDK expression in G1 cells. We observed that DDK expression in G1 cells lead to premature phosphorylation of Sae2, and by performing DNA end resection and HR assays we observed that synthetic activation of DDK in G1 leads to a moderate activation of HR, highlighting the central role of DDK in DSB repair pathway choice. Cell cycle kinases also regulate the resolution of recombination structures by the Mus81- Mms4 nuclease during late steps of HR. DDK and Cdc5 can physically interact with each other and it was previously shown that this two-kinase complex is required for phosphorylation and activation of Mus81-Mms4. In a second project we therefore focused on the DDK-Cdc5 kinase complex and how it works as two-kinase complex to phosphorylate Mus81-Mms4 and other proteins. In a candidate approach, we identified a novel phosphorylation substrate of the kinase complex, the DNA replication factor Sld2, suggesting the DDK-Cdc5 complex might be a more general regulator of M phase. We developed protocols to purify to homogeneity from yeast cells the DDK-Cdc5 complex and the single kinases. Through a series of in vitro experiments, we showed that the DDK- Cdc5 complex was overall active as well as the single kinases. Different phosphorylation substrates were specifically phosphorylated by either one of the two kinases, either when on their own or as part of the complex, suggesting that within the complex each of the kinases could act as scaffold or adaptor for substrates. Lastly, we showed that also the human proteins DDK and PLK1 (human ortholog of Cdc5) physically interact, indicating that the DDK-Cdc5/DDK-PLK1 complex is an evolutionary conserved composite cell cycle regulator. Taken together, the work presented in this thesis uncover a novel role of DDK in regulating DSB repair and offer insights into the evolutionary conserved DDK-Cdc5 kinase complex.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 11. Januar 2022
• 1. Bericht­erstatter:in: Becker, Peter