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Identification of the Prenucleosome and Analysis of the Mechanism of ATP-Dependent Chromatin Assembly /

  • Author(s): Torigoe, Sharon E.
  • et al.
Abstract

The assembly of chromatin is a necessary process that must occur following DNA-utilizing processes, such as replication, repair, and transcription. Chromatin is a complex of DNA and proteins that is responsible for packaging and organizing DNA into the eukaryotic nucleus and also serves as a regulator of all DNA-utilizing processes. The basic repeating unit of chromatin is the nucleosome, which consists of approximately 147 base pairs of DNA wrapped around a histone octamer core. Chromatin assembly is facilitated by core histone chaperones and ATP -dependent motor proteins. For this dissertation, I have investigated the mechanism of chromatin assembly using a purified, defined in vitro chromatin assembly system, consisting of relaxed DNA, ATP, core histones, the histone chaperone NAP1, and the ATP-driven motor proteins ACF or CHD1. A novel nonnucleosomal histone-DNA complex, termed the prenucleosome, was identified as an intermediate of chromatin assembly. Whereas this intermediate of chromatin assembly is biochemically distinct from canonical nucleosomes, the prenucleosome is indistinguishable from a nucleosome by atomic force microscopy. Analysis of chromatin remodeling-defective mutants of CHD1 demonstrated that ATP-dependent chromatin assembly is a distinct activity from chromatin remodeling. These findings indicate a multi-step mechanism for chromatin assembly. Histone chaperones first deposit histones onto DNA to generate prenucleosomes. These prenucleosomes are subsequently converted into randomly-positioned canonical nucleosomes by ATP-dependent motor proteins. Lastly, ATP- driven motor proteins reposition these nucleosomes into periodic arrays. Altogether, these studies provide new insight into the mechanism of ATP-dependent chromatin assembly, highlighting the necessity of ATP-driven motor proteins in this process

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