RESEARCH
Chromatin plays important regulatory roles in all steps of DNA replication, by dictating origin start-site selection and modulating replication fork progression. Only by studying chromatin replication, we argue, will we understand the molecular basis of genome propagation. To this end, we have developed new protocols to perform visual biochemistry experiments under the cryoelectron microscope, to image chromatin duplication at high resolution, frozen as it is being catalysed. Using these strategies, we want to generate a molecular movie of the entire replication reaction. Our achievements will change the way we think about genome stability in eukaryotic cells.
CHROMATIN REPLICATION
The natural substrate of the eukaryotic replisome is chromatin and not just DNA.
During replication, parental histones must be disassembled ahead of the replication fork and redeposited onto duplicated DNA. We plan on imaging reconstituted chromatin replication reaction to understand how parental histones are redistributed at the replication fork.
To achieve this goal we will first need to characterise DNA replication origin activation under the electron microscope.
HELICASE LOADING
Replicative helicases unwind DNA exposing the single-stranded template for the replicative polymerases. The MCM helicase motor is loaded onto duplex DNA as an inactive double hexamer in a process that requires an ATPase assembly named ORC and loading factors Cdc6 and Cdt1. Two models exist for how a double hexamer is formed. One model involves the sequential loading of two helicases mediated by one lone ORC complex. According to a second model, two ORC assemblies symmetrically load two MCM helicases to form the double hexamer. We want to use high-resolution single-particle cryo-EM to visualise the entire helicase loading reaction as it occurs in solution.
ORIGIN ACTIVATION
The DNA untwisting function in MCM is switched on upon ATP binding and the concomitant recruitment of helicase activators GINS/Cdc45 (together forming the CMG). We want to use cryo-EM to establish whether DNA untwisting involves impairing of nitrogenous bases and whether both strands remain trapped inside the MCM ring at this stage. Recruitment of firing factor Mcm10 is understood to cause an isomerization in the CMG helicase with opening of MCM and ejection of the lagging-strand template. At this stage CMG can start hydrolyzing ATP and translocate on the leading-strand template. We want to understand strand ejection and ATPase driven DNA translocation using time-resolved cryo-EM.
THE REPLISOME
Genome duplication requires tight coordination between parental duplex-DNA unwinding and daughter-strand synthesis within the replisome, to prevent the accumulation of vulnerable single-stranded DNA segments and the onset of genomic instability.
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We are interested in understanding replisome biogenesis and the mechanism of coupled DNA unwinding and synthesis at the replication fork.
TECHNIQUE DEVELOPMENT
We aspire to perform biochemical experiments under the cryo-electron microscope.
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The system we use is highly complex, involving sequential ATP hydrolysis, post-translational modification and nucleotide switch and incorporation events.
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We are devising new biochemical tools to minimise compositional heterogeneity in our sample, as well as computational methods to reconstitute replication reactions in silico.
COLLABORATIVE WORK
Working together with Peter Cherepanov at the Crick, we are investigating the molecular basis of retroviral intergration into host-cell chromosomes.
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We have determined the first subnanometre resolution structure of an enzyme bound to a nucleosome and we now want to establish how retroviral integration affects nucleosome repositioning.