DDK shapes origin chromatin structure and replication efficiency by finetuning INO80 function

Bansal, Priyanka (2024): DDK shapes origin chromatin structure and replication efficiency by finetuning INO80 function. Dissertation, LMU München: Medizinische Fakultät

Abstract

Eukaryotic genomic DNA is wrapped around histone proteins to form nucleosomes, the basic units of chromatin. Chromatin is a higher-order structure and key regulatory element for DNA-template processes. Nucleosomes can regulate the access to DNA during replication, transcription, recombination, and repair. Any deregulation of these processes can cause genomic instability and can result in diseases like cancer. Hence, it is crucial to gain insights into the fundamental mechanisms that regulate nucleosome dynamics. Replication initiates at specific DNA sequences called origins of replication. In the budding yeast Saccharomyces cerevisiae (S. cerevisiae), origins are nucleosome-free regions (NFRs) flanked by arrays of well-positioned nucleosomes. This specific chromatin structure helps in origin selection and pre-replication complex assembly at origins. At the beginning of the S phase of the cell cycle, the pre-replication complex is converted into the active replication machinery by DDK (Dbf4-dependent kinase). The active replication machinery encounters nucleosomes as a barrier and therefore requires assistance of chromatin factors (ATP-dependent chromatin remodelers and histone chaperones). Upon committing to cell division, in G1 phase, chromatin factors interact with origin recognition complex (ORC) to form arrays of well positioned nucleosomes. However, how these chromatin factors are regulated throughout the cell cycle is unknown. Because low levels of DDK have been observed at early origins during G1 phase of the cell cycle, we speculated that DDK might be involved in the regulation of chromatin factors at early origins of replication, possibly by direct phosphorylation. In this study, we utilized S. cerevisiae to discover phosphorylation events by DDK with an emphasis on chromatin factors to understand the DDK phosphorylation network in the nucleus. Using a mass spectrometry omics approach, we found DDK phosphorylation events on many nuclear proteins. In particular, we identified the Arp8 subunit of INO80 to be phosphorylated by DDK. We found that Arp8 phosphorylation does not peak in S phase, but appears during G1, consistent with a role of DDK in phosphorylating and regulating INO80 function prior to S phase. INO80 is a multi-subunit ATP-dependent chromatin remodeler and a known player of chromatin replication. We dissected the functional relevance of DDK-dependent phosphorylation on the INO80 complex. We showed that DDK phosphorylation of Arp8 is essential for the integrity of INO80, in particular for the interaction of Ino80 with the NHP10 and ARP8 modules. When Arp8 was not phosphorylated, it leads to defects in DNA replication, structural changes within the INO80 complex which reduced ATP hydrolysis and generated incorrect nucleosomal spacing with larger linker length. In the model, we propose that during G1 phase, INO80 and DDK interact with ORC subunits at early origins of replication where DDK phosphorylates Arp8 subunit of INO80 for precise nucleosomes positioning. Overall, our study provides a comprehensive resource of nuclear phosphorylation events that are regulated by DDK. Our study further establishes a model where INO80 activity to accurately position nucleosomes around origins of replication is regulated by direct phosphorylation of DDK prior to the onset of S phase replication. To our knowledge, this is the first example of any organism, where the nucleosome spacing activity of a chromatin remodeler is directly regulated by signals of the cell cycle machinery.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 1. Oktober 2024
• 1. Bericht­erstatter:in: Imhof, Axel