The role of Orc6 in ORC binding-site switching during helicase loading

Abstract

DNA replication is a fundamental cellular process that enables proper maintenance of genomic integrity and cellular identity as cells divide. Initiation of bidirectional DNA replication is a key requirement for complete duplication of the genome. The first step in eukaryotic DNA replication is origin licensing, during which the origin recognition complex (ORC) coordinates loading of two Mcm2-7 hexameric helicases in opposite orientations to form the Mcm2-7 double hexamer. These Mcm2-7 double hexamers mark sites of eventual bidirectional replication initiation upon entering S-phase, when activation of these helicases and recruitment of DNA polymerases leads to new DNA synthesis. In. S. cerevisiae, ORC initially binds to a high affinity site at origins of replication to load the first Mcm2-7 complex. ORC is then able to bind a secondary inverted binding site at origins to load a second Mcm2-7 in the opposite orientation to coordinate formation of the Mcm2-7 double hexamer. Previous single-molecule studies show that one ORC can recruit and load both Mcm2-7 helicases at origins. This one-ORC helicase-loading model requires a dramatic change in ORC location to transition between its primary and secondary binding site. Importantly, this must occur without release of ORC from the site of helicase loading. How this binding-site switch is mediated is currently unknown. In this thesis, we used single-molecule Förster resonance energy transfer (smFRET) assays to study interactions between ORC and Mcm2-7 during helicase loading. We found that upon recruitment of Mcm2-7 to origin DNA, in addition to the previously described ORC C-terminal tier interaction with Mcm2-7 C-terminal tier, Orc6N forms an interaction with the N-terminal tier of Mcm2-7 (Mcm2-7N). We identified elements of Orc6 required to mediate this interaction, as well as potential mechanisms of inhibition of this interaction by the regulatory kinase CDK. The kinetics of this interaction indicate that Orc6N interacts with Mcm2-7N well before ORC undergoes its binding site switch, consistent with a role of this interaction in retaining ORC at the site of helicase loading during this transition. Additionally, we demonstrate a role for Orc6 in mediating stable second Mcm2-7 recruitment. These findings emphasize the role of protein-protein interactions in enabling ORC to coordinate loading of oppositely-oriented Mcm2-7 helicases to enable bidirectional replication and identify new functions for Orc6 during this process.