SINGLE MOLECULE STUDIES OF NUCLEOSOME INHERITANCE

Abstract

Eukaryotic gene expression is tightly coupled to the spatial organization and modification of nucleosomes. During DNA replication, parental nucleosomes are partitioned approximately equally between the nascent DNA strands where they serve as a template for modification of nascent histones. Replication fork progression is tightly coupled to the deposition of nascent histones behind the replication fork, however the deposition of parental nucleosomes is poorly understood. Reconstitution of eukaryotic replication will be essential to dissecting the underlying mechanisms responsible for the coordination of replication and re-chromatinization of the genome. Pairing in vitro approaches with single-cell manipulation has the potential to contextualize the in vitro findings with in the complexities of the cellular environment. Here I present three distinct projects that lay the foundation towards the goal of ascertaining the fundamental mechanisms the drive chromatin replication and epigenetic inheritance. First is the characterization of the a passive-nucleosome transfer mechanism in the context of a simplified replication system. Second, the preliminary experiments aimed at characterizing the eukaryotic replicative helicase and reconstituting the eukaryotic replisome. Finally, a single-cell injection technique used to study asymmetric cell division in the C. elegans embryo.