Periodic Reporting for period 4 - CHROMOREP (Reconstitution of Chromosome Replication and Epigenetic Inheritance)
Berichtszeitraum: 2020-05-01 bis 2022-04-30
In addition to providing a deep understanding of a fundamentally important cellular process, our work may have important implications for understanding the aetiology of human cancer. It has emerged in recent years that most and perhaps all cancers exhibit some form of ‘replicative stress’, loosely defined as perturbation of normal replication rates and accumulation of DNA damage during replication. It is unclear whether there is a single cause of replicative stress or whether replicative stress is an umbrella term for a variety of biochemical mistakes, which may be different in different cancers. Our work will help understand the biochemical mechanism by which chromosomes are replicated. This work will synergise with other work from my lab aimed at understanding replicative pathways which are misregulated in cancer.
The aim of our project is to understand how replication forks contend with nucleosomes and how gene expression patterns are re-established during and after DNA replication using a ‘bottom-up’ approach to reconstitute this process with purified proteins, based on a DNA replication system developed in our laboratory.
I. Origin choice and timing. All eukaryotic cells initiate replication from multiple ‘replication origins’ distributed along chromosomes which each have a signature efficiency and time of replication during S phase. This project is aimed at understanding how nucleosome arrays (chromatin) affect the choice of replication origins and the time at which origins are activated during S phase.
1. Origin choice – We have recently published work showing that chromatin enforces replication origin selection (Kurat et al. Mol. Cell 2017). We have obtained atomic resolution understanding of how the MCM helicase is loaded at replicartion origins and how it is activated in S phase.
2. Origin timing – We have established a competition assay in which plasmids containing different histone modifications are mixed together and replicated to begin to understand how early firing origins out-compete late origins for limiting replication factors.
II. Mechanisms of nucleosome displacement and potential transfer to daughter chromatids during DNA replication
1. Displacement of parental nucleosomes – We have published work showing histone ‘chaperones’ (proteins that bind histones and neutralise their charge), nucleosome remodellers (enzymes that use ATP hydrolysis to push nucleosomes around) and histone acetylation all contribute to rapid replication through chromatin (Kurat et al. Mol. Cell 2017). This work also showed parental nucleosomes were efficiently transferred behind the progressing fork. We have reconstituted the pathway by which newly synthesised histones are incorporated into replicating DNA to complement this work.
2. Pathway(s) for inheriting parental nucleosomes – We have combined the de novo nucleosome assembly pathway with the chromatin replication system to acieve complete chromatin replication. We are using this to understand competition between the two pathways.
III. Inheritance of chromatin states during and after DNA
1. Recruitment of histone modifiers during DNA replication – We have established an approach to identify modifiers recruited to DNA in extracts and have identified several of these.
2. Coupling chromatin replication to transcriptional silencing – We have reconstituted replication of heterochromatin. We have shown that FACT, which is sufficient to promote replication of euchromatin, is not sufficient to promote replication of heterochromatin. Our current dtaa suggests transient histone acetylation followed by SIR2-dependent deacetylation is sufficient to promote replication through heterochromatin.