Role of Rtt106 in replication coupled nucleosome assembly

Nucleosomes, the repeating units of chromatin, are made up of core histones each containing two heterodimers of H2A-H2B and a heterotetramer of H3-H4. Deposition of H3-H4 onto DNA is a key step in nucleosome formation. Deposition of H3-H4 is mediated by a group of proteins called histone chaperones that binds H3-H4 and promotes nucleosome formation. The transfer of parental histones containing epigenetic marks to nascent DNA is thought to occur in a process coupled to DNA replication known as replication-dependent chromatin assembly. Chromatin assembly factor 1 (CAF-1), anti-silencing factor 1 (Asf1) and Rtt106 are H3-H4 chaperones in budding yeast. While, Asf1 binds a H3-H4 dimer through the same H3 interface involved in formation of H3-H4 tetramer, it is not known how CAF-1 and Rtt106 binds H3-H4. Therefore, determining the molecular form (dimer or tetramer) of H3-H4 that is bound to histone chaperones would provide important insight into mechanism of replication dependent nucleosome assembly.;The goal of my studies was to determine if the histone chaperone Rtt106 binds to an H3-H4 dimer or tetramer during DNA replication dependent nucleosome assembly. Using a sequential affinity purification procedure of epitope tagged proteins and H3 lysine 56 acetylation dependent binding of Rtt106 to histone H3-H4, I determined that Rtt106 binds to H3-H4 tetramers in vivo. In addition, utilizing gel filtration chromatography and tandem affinity purification of epitope tagged Wild type and truncated and point mutant Rtt106, I have shown that Rtt106 exists as a homo-oligomer in vivo and in vitro and that oligomerization is important for Rtt106 to bind H3-H4. Similarly, using a florescent silencing assay and exposure to the genotoxic agent CPT, I have shown that Rtt106 plays a role in maintaining heterochromatin silencing and genomic integrity. Together, these studies reveal a novel mode of interaction between a H3-H4 chaperone and H3-H4 and indicate that a H3-H4 tetramer can be deposited intact for nucleosome formation.