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Mechanisms of human DNA replication

Project description

Initiation of DNA replication unveiled

DNA replication is vital for cell propagation and is initiated at various sites throughout the genome. The process involves several proteins with precise functions that deliver replication accuracy. Although several studies have shed light on DNA replication initiation in mammalian cells, the process in human cells is incompletely understood. The scope of the EU-funded Human Repl Mech project is to investigate DNA replication initiation and regulation. Researchers will use molecular biology and biochemical techniques to reconstitute human DNA replication in vitro and study the role of helicase, the enzyme responsible for unwinding the DNA double helix. Results will provide unprecedented knowledge in one of life’s most fundamental processes and pave the way towards the design of antiproliferative drugs.

Objective

Eukaryotic DNA replication is mediated by a complex machinery consisting of several dozen proteins that ensures precise duplication of chromosomes in S phase of the cell cycle. The Diffley laboratory recently reconstituted the core Saccharomyces cerevisiae replication machinery in vitro from 19 replication factors containing about 50 individual protein subunits. This system allowed to study mechanisms of yeast MCM loading onto DNA and of yeast CMG replicative helicase activation. In human cells however, the process of replication initiation and its regulation are less well understood. In particular, the firing factors that activate the CMG helicase differ in domain composition and phospho-regulation from the yeast orthologs. Mechanistic studies are required to understand how human DNA replication is initiated and regulated at the molecular level. I will first reconstitute human DNA replication in vitro from completely recombinant components using methodology that I developed during my PhD. I will then use this system to study how the human replicative helicase is activated on the molecular level and how activation is regulated by phosphorylation using biochemistry. I will use cryo-electron microscopy (cryo-EM) to study the architecture of complexes involved in replication initiation. The reconstituted human system could in future enable studies on disease mechanisms and could allow screening for antiproliferative small molecule inhibitors.

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Coordinator

THE FRANCIS CRICK INSTITUTE LIMITED
Net EU contribution
€ 212 933,76
Address
1 midland road
NW1 1AT London
United Kingdom

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Region
London Inner London — West Camden and City of London
Activity type
Research Organisations
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Other funding
€ 0,00