| The information encoded in all eukaryotic genomes is organized into a nucleoprotein complex called chromatin. The fundamental repeating unit of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around an octamer of histone proteins. Chromatin is critical for regulating gene expression and segregating the genome from one generation to the next. As cells replicate their DNA they must also repackage it into chromatin. It is essential for viability that nucleosome assembly and DNA synthesis occur in concert. My doctoral research has been to investigate the molecular mechanisms governing this coordination, both in the budding yeast Saccharomyces cerevisiae and in human cells.; Two conserved histone chaperones, Chromatin Assembly Factor-1 (CAF-1) and Asf1, have been implicated in replication-coupled chromatin assembly. In one component of my thesis work, I demonstrated that CAF-1 is essential for completing DNA replication in human cells. The main focus of my thesis work, however, has been Asf1. Biochemical and genetic data suggest that CAF-1 and Asf1 cooperate to deliver (H3/H4)2 tetramers to replicating DNA. CAF-1 is recruited to sites of DNA synthesis through an interaction with the DNA polymerase processivity protein, PCNA. However, the molecular mechanisms connecting Asf1 to DNA replication were unknown.; Chapters Two and Three describe my discovery that Asf1 binds directly to a component of the replication apparatus, Replication Factor C (RFC). RFC is the multi-subunit enzyme responsible for loading and unloading PCNA at sites of DNA synthesis. Asf1 inhibits RFC activity in a manner regulated by histones and primed DNA. These data suggest that Asf1 acts as a sensor that couples histone deposition and DNA synthesis via regulation of RFC. Additionally, my studies uncovered a new function for Asf1 during DNA replication; Asf1 promotes the stability of stalled replication forks, and I hypothesize this activity is a consequence of the ability of Asf1 to modulate RFC activity. Together, my studies demonstrate that replication-coupled histone deposition is essential for completing DNA synthesis, and regulated coupling of these two processes occurs via the histone sensor Asf1. |
