Single-molecule studies of eukaryotic helicase loading

Abstract

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis.Includes bibliographical references.Cells must duplicate their genomic content fully and accurately in each cell cycle to maintain cellular identity and ensure the viability of their progeny. The first step in eukaryotic DNA replication initiation is the loading of the heterohexameric Mcm2-7 helicase. In eukaryotes helicase loading is tightly regulated to ensure that this event only occurs during the GI phase of the cell cycle. Because helicase activation can only occur once cells enter S phase, cells can only load and activate the helicase once per cell cycle, ensuring the genome is replicated once and only once in each cell cycle. The selection of sites of helicase-loading also marks all potential origins of replication. Once loaded, the helicases encircle dsDNA and are linked in a head-to-head double hexamer. Although the proteins involved in helicase loading are known (ORC, Cdc6, and Cdt 1), the mechanism by which they load two oppositely-oriented helicases and ensure their proper architecture remains under intense investigation. In this thesis I describe a novel single-molecule helicase-loading assay that allows monitoring of protein associations and dissociations on a one-second time scale. By labeling pairs of helicase-loading proteins simultaneously, I determined the relative time of association and dissociation for the helicase and each of the helicase-loading proteins during helicase loading. Additionally, I determined the stoichiometry of each helicase-loading protein with respect to the origin DNA. Adapting this assay to read out distance information using single-molecule FRET, I monitored formation of the final double-hexamer in real time. These single-molecule assays uncovered that helicase loading occurs in a one-at-a- time manner and discovered novel steps in the mechanism of helicase loading. Following the initial association of ORC/Cdc6/Cdtl/Mcm2-7 with the origins of DNA replication, Cdc6 and then Cdtl are released sequentially. A new Cdc6, and Cdtl/Mcm2- 7 are subsequently recruited and the same ordered sequential release is observed, although with different kinetics. Although two Cdc6 and Cdtl proteins are required for loading a double hexamer, a single ORC is sufficient. Additionally, double-hexamer formation is a rapid event upon association of the second helicase, suggesting a model in which the two helicases are recruited and loaded around dsDNA by distinct mechanisms.by Simina Ticau.Ph. D