The role of FACT in nucleosome inheritance
File(s)
Author(s)
Smith, Christopher Kevin
Type
Thesis
Abstract
Nucleosomes are a barrier to the eukaryotic replisome and so must be at least partially disassembled. However, parental nucleosomes may possess important post-translational modifications that regulate local gene expression. They must, therefore, be redeposited onto the daughter DNA templates at the precise genomic location from which they were removed. How parental histones are transferred to the daughter DNA templates is currently unclear. The Saccharomyces cerevisiae histone chaperone FACT (Facilitates chromosome transactions) is required for the replication of chromatin in vitro and is a component of the replisome progression complex (RPC) in vivo.
Using a pulldown approach with purified proteins, I confirmed that FACT directly binds DNA polymerase alpha and found that FACT also directly binds Csm3/Tof1 and the catalytic domain of DNA polymerase epsilon. I additionally confirmed that FACT binds the Mcm2-7 complex in the presence of histones and found that FACT also binds Ctf4 in the presence of histones. Using crosslinking mass spectrometry and alanine scanning mutagenesis, I identified the residues within Tof1 and Pol2 that directly bind FACT. I also identified the residues within Ctf4 that bind specifically to histones H3-H4.
Therefore, Csm3/Tof1 may incorporate FACT into the RPC to facilitate parental nucleosome disassembly. FACTs direct interaction with Pol2 may facilitate the transfer of parental histones to DNA polymerase epsilon for histone deposition onto the leading strand. FACTs histone-mediated interactions with Mcm2 and Ctf4 may facilitate the transfer of parental histones to DNA polymerase alpha for histone deposition onto the lagging strand. The disruption of each interaction with FACT may reveal how parental histones, and thus epigenetic information, can be faithfully inherited in vivo.
Using a pulldown approach with purified proteins, I confirmed that FACT directly binds DNA polymerase alpha and found that FACT also directly binds Csm3/Tof1 and the catalytic domain of DNA polymerase epsilon. I additionally confirmed that FACT binds the Mcm2-7 complex in the presence of histones and found that FACT also binds Ctf4 in the presence of histones. Using crosslinking mass spectrometry and alanine scanning mutagenesis, I identified the residues within Tof1 and Pol2 that directly bind FACT. I also identified the residues within Ctf4 that bind specifically to histones H3-H4.
Therefore, Csm3/Tof1 may incorporate FACT into the RPC to facilitate parental nucleosome disassembly. FACTs direct interaction with Pol2 may facilitate the transfer of parental histones to DNA polymerase epsilon for histone deposition onto the leading strand. FACTs histone-mediated interactions with Mcm2 and Ctf4 may facilitate the transfer of parental histones to DNA polymerase alpha for histone deposition onto the lagging strand. The disruption of each interaction with FACT may reveal how parental histones, and thus epigenetic information, can be faithfully inherited in vivo.
Version
Open Access
Date Issued
2021-11
Date Awarded
2022-07
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Diffley, John
Alcaide, Luis
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)