Establishment and function of nucleosome organization at eukaryotic chromosome replication origins

Chacin, Erika (2023): Establishment and function of nucleosome organization at eukaryotic chromosome replication origins. Dissertation, LMU München: Medizinische Fakultät

Abstract

The whole genome has to be replicated only once per cell cycle and only during S phase. DNA replication initiates at specific sequences within the genome referred as origins of replication, and the number of origins correlates with the size of the genome. Problems with replication are associated with genomic instability and replication stress, both hallmarks of cancer and/or growth defects. The budding yeast S. cerevisiae has been the preferred model organism to study DNA replication in detail because (a) its origins are well defined, (b) decades of genetics have painted a pretty clear picture of the process, and importantly, (c) replication has been reconstituted with purified components. Replication origins contain an ARS that harbors an AT-rich conserved motif, known as the ACS, which is only present in yeast. ORC binds specifically to this motif and, together with other loading factors, recruits and loads the replication helicase, the MCM or Mcm2-7/Cdt1, as a double-hexamer. The chromatin structure at yeast origins is characterized by an NFR with flanking nucleosomal arrays of regular spacing. ORC has been shown to influence nucleosome positioning at origins of replication. However, prior to our work, the precise mechanism was unknown. Further, it was unclear if this stereotypical chromatin structure is functionally important for replication. By screening ORC and seventeen purified chromatin factors via genome-scale in vitro reconstitution, we were able to determine the factors that establish this chromatin structure at origins. We found that ORC works together with the spacing remodelers INO80, ISW1a, ISW2 and Chd1 to generate nucleosome arrays at origins. Moreover, by testing different mutations of the Orc1 subunit of ORC, we were able to dissect ORC’s chromatin function at origins of replication by uncoupling it from its canonical function as the MCM loader. We found that nucleosome array generation depends on Orc1’s ability to hydrolyze ATP and on the BAH and IDR domains. These mutations were lethal in vivo and lost their arrays in vitro. Most importantly, this hindered DNA replication in in vivo and in vitro. To replicate chromatinized DNA, the replisome requires the assistance of chromatin factors. It is known that some chromatin remodelers and histone chaperones enhance replication rates. In this context, we characterized Yta7 as a new type of chromatin remodeler namely chromatin segregase, that is different from the classical SF2 chromatin remodelers. One major difference is that its motor subunit belongs to the AAA+-ATPase superfamily. We found that Yta7 is activated during the S phase by S-CDK, which phosphorylates it in close proximity of the ATPase domain. Interestingly, phosphorylation causes stimulation of the ATPase activity, activation of its chromatin segregase function and strongly facilitated chromatin replication in vitro. Finally, we collaborated with the Duderstadt lab at the MPI Biochemistry, to study how FACT, a two-subunit complex composed of Spt16 (SuPpressor of Ty’s 16) and Pob3 (Pol1 Binding 3), engages with the replisome to enhance replication as previously reported. We found that Spt16’s N-terminus is required for FACT’s direct interaction with the replication machinery while the C-terminus of both Spt16 and Pob3 is required for nucleosome interaction ahead of the replication fork.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Origins of replication, chromatin replication, nucleosomes, origin recognition complex, chromatin remodelers, nucleosome arrays, in vitro reconstitution, yeast
• 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: 8. Dezember 2023
• 1. Bericht­erstatter:in: Korber, Philipp