Wednesday, 20 April 2022 | 12.00 noon – ICGEB Trieste, ITALY
Assistant Professor, Bioscience, Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, SAUDI ARABIA
Human DNA replication under the microscope: Visualizing the lagging strand replisome at high-resolution using cryo-EM
(Host: L. Banks)
Cryogenic electron microscopy (cryo-EM) allows to visualize intact molecular machines at high-resolution and has revolutionized the field of structural biology. In our laboratory, we employ single-particle cryo-EM to decipher the fundamental processes operating in human DNA replication. In this talk, I will present the methods for in vitro reconstitution, freezing and structure determination of the replisome components that duplicate the DNA lagging strand. Lagging strand replication requires the action of three enzymes, DNA polymerase δ, Flap endonuclease 1 and Ligase 1, which are coordinated by the sliding clamp PCNA to execute the three central steps of the process: Okazaki fragment gap filling, flap cleavage and fragment nick sealing. The structural elucidation of these holoenzymes explains how PCNA acts as a “toolbelt” where each enzyme is recruited at the correct step to perform the corresponding DNA transaction, and has allowed the mapping of several known disease-causing mutations. Finally, I will delineate how cryo-EM can be combined with other single-molecule techniques to characterize the dynamic communication between proteins working in complex biomolecular systems such as the replisome.