Periodic Reporting for period 2 - MeChroRep (Mechanism of Chromatin Replication)
Berichtszeitraum: 2023-11-01 bis 2025-04-30
Before a human cell divides, it must make an exact copy of each of its 46 chromosomes. This involves replicating more than 6 billion base pairs of DNA precisely once; every sequence must be replicated but no sequence can be replicated more than once. In addition, all of the proteins associated with that DNA must also be duplicated and bound at the same locations on both daughter chromosomes. The precise duplication of this chromatin — genomic DNA with all its associated proteins — underpins stable passage of genetic and epigenetic information through the cell cycle. During DNA replication, every base pair must be unwound, and every bound protein transiently displaced. Yet gene expression patterns are re-established within minutes of DNA replication indicating that duplication of the underlying chromatin structure must be rapid. Errors in these processes can lead to developmental defects and diseases including cancer. The overarching aim of the MeChroRep project is to generate a deep understanding of how DNA replication is coordinated with the disassembly and reassembly of chromatin to ensure accurate chromosome duplication
Much of the work forming the background to this application was based on the reconstitution of complete DNA replication with purified yeast proteins. This system is extremely powerful for obtaining detailed molecular understanding of this process, but, because it uses yeast proteins, its relevance for human replication is unclear. To address this we have begun to reconstitute DNA replication with purified human proteins. This past year we described the reconstitution of the first step -- loading of the human MCM replicative DNA helicase.
We are in the process of analysing how the essential histone chaperone, FACT, and a variety of histone binding domains in replisome components contribute to both disruption of nucleosomes ahead of the replication fork and redposition of parental histones into nucleosomes behind the fork. We are also in the process of analysing how re-establishment of parental histones into chromatin is coordinated with deposition of newly synthesised histones into chromatin.
We are in the process of analysing how the essential histone chaperone, FACT, and a variety of histone binding domains in replisome components contribute to both disruption of nucleosomes ahead of the replication fork and redposition of parental histones into nucleosomes behind the fork. We are also in the process of analysing how re-establishment of parental histones into chromatin is coordinated with deposition of newly synthesised histones into chromatin.
Aim 1: We will continue our biochemical analysis of the roles of the histone chaperone FACT and the histone binding domains in replisome components. When complete, this work will provide a detailed understanding of how the replisome disassembles the highly stable nuclesomes during DNA replication
Aim 2: We will use our biochemical approaches together with novel sequencing technologies to fully understand how the histones from nucleosomes ahead of thr replication fork are efficiently distributed to the nascent leading and lagging strands behind the replisome..
Aim 3: Because there is twice as much DNA after replication the parental histones can make up only 50% of the final chromatin. Therefore, this parental histone inheritance pathway must be coordinated with the deposition of newly synthesised histones. We have reconstituted this de novo pathway and are examining how it interacts with the parental inheritance pathway.
Aim 4: In addition to histones, other chromatin-associated proteins must also be inherited during replication. Of particular importance are heterochrmatin-associated facors. We have reconstituted yeast heterochromatin with histones and the Sir2-4 proteins and we will analyse how the Sir proteins are inherited during replication. This should provide a detailed understanding of how heterochromatic states are preserved during DNA replication.
Aim 5: We will ultimately establish analogous systems to study these processes with human proteins. We have reconstituted the first step in replication and will hopefully have reconstituted the entire chromatin replication reaction by the end of this funding period.
Aim 2: We will use our biochemical approaches together with novel sequencing technologies to fully understand how the histones from nucleosomes ahead of thr replication fork are efficiently distributed to the nascent leading and lagging strands behind the replisome..
Aim 3: Because there is twice as much DNA after replication the parental histones can make up only 50% of the final chromatin. Therefore, this parental histone inheritance pathway must be coordinated with the deposition of newly synthesised histones. We have reconstituted this de novo pathway and are examining how it interacts with the parental inheritance pathway.
Aim 4: In addition to histones, other chromatin-associated proteins must also be inherited during replication. Of particular importance are heterochrmatin-associated facors. We have reconstituted yeast heterochromatin with histones and the Sir2-4 proteins and we will analyse how the Sir proteins are inherited during replication. This should provide a detailed understanding of how heterochromatic states are preserved during DNA replication.
Aim 5: We will ultimately establish analogous systems to study these processes with human proteins. We have reconstituted the first step in replication and will hopefully have reconstituted the entire chromatin replication reaction by the end of this funding period.