Alessandro Costa: Projects

We study the structure and function of macromolecular machines that preserve chromosome integrity, with a particular focus on chromosome duplication. DNA replication onset and progression require the orchestrated action of multiple enzymes that often function as large, modular assemblies.

To describe the architecture and dynamics of the switches and motors that drive DNA replication, we combine single-particle cryo-electron microscopy (EM), molecular modelling, and biochemistry aimed at elucidating the mechanics of replication origin activation and fork progression.

DNA replication - structure of the translocating eukaryotic replicative helicase

The Cdc45/Mcm2-7/GINS (CMG) helicase unwinds the DNA double helix during replication in eukaryotes. How the CMG is assembled and how it engages DNA substrates remains only partially understood. Using negative-stain electron microscopy, we have determined the structure of the CMG in the presence of a slowly hydrolysable ATP analogue and a DNA duplex substrate with a 3' single-stranded tail. The structure shows that the Mcm motor subunits of the CMG bind single- and not double-stranded DNA, supporting a steric exclusion mechanism for replication fork unwinding.

We used biotin-streptavidin labelling to establish the polarity by which DNA enters into the Mcm2-7 channel, and elucidate how Cdc45 keeps the helicase topologically linked to the translocation strand during DNA unwinding. The Mcm2-7 motor subcomplex forms a right-handed spiral when DNA-bound, revealing unexpected similarities between the CMG and other hexameric ATPases such as the bacterial DnaB helicase and the Rpt1-6 AAA+ motor of the eukaryotic proteasome.

We identified a subpopulation of dimeric CMGs, which allowed us to establish the subunit register of Mcm2-7 double hexamers assembled onto DNA before replication origin firing.

Altogether, our results provide novel important insights into the nucleoprotein architecture of the replication fork.

We are now interested in describing the detailed molecular mechanism of DNA translocation by the activated Mcm2-7 AAA+ motor. To this end, we have optimised preparations of substrate-bound CMG molecules embedded in vitreous ice for high-resolution cryo-electron microscopy.

By imaging our nucleoprotein preparations using a direct electron detector on a 300 kV EM instrument, we aim at determining the near-atomic resolution structure of the translocating CMG helicase in various stages of the ATP hydrolysis cycle.

Our results will inform us on the mechanism of ATPase cycling and nucleic acid translocation by a hetero-hexameric motor.

DNA replication - helicase/polymerase coupling

Genome duplication requires tight coordination between parental duplex-DNA unwinding and daughter-strand synthesis within the replication machinery, to prevent the accumulation of vulnerable single-stranded DNA segments and the onset of genomic instability.

We recently employed single-particle electron microscopy coupled with biochemistry and crystallography (in collaboration with Luca Pellegrini at the University of Cambridge), to describe the architecture of the Ctf4 'helicase-polymerase bridging factor', either alone or bound to components of the CMG helicase and the DNA Polymerase alpha/primase assemblies.

We showed that budding yeast Ctf4 forms a homo-trimericdisk, suggesting that it has the ability to link multiple factors at replication forks. Indeed, the Ctf4trimer contains three docking sites that recognize a conserved motif mapping within in the Pol alpha catalytic subunit as well as one of the four GINS subunits of the CMG helicase.

Importantly, we showed that the Ctf4 trimer is capable of simultaneously binding to GINS and the amino terminus of the Pol alpha catalytic subunit. These findings establish the architectural framework for further mechanistic studies of the elongation step of DNA replication in eukaryotic cells.

Alessandro Costa

Alessandro Costa

alessandro.costa@crick.ac.uk
+44 (0)20 379 61812

  • Qualifications and history
  • 2007 PhD, Imperial College London, UK
  • 2007 Postdoctoral fellow, University of Oxford, UK
  • 2009 EMBO Postdoctoral fellow, University of California Berkeley, USA
  • 2012 Establish lab at the London Research Institute, Cancer Research UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK