License to Replicate: Elucidating the Molecular Mechanisms of Metazoan DNA Replication Origin Licensing

Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Bleichert, Franziska

Abstract

DNA replication initiation is a tightly regulated process that requires the coordinated assembly of replication machineries throughout the genome. During the first step of initiation called origin licensing, two copies of the MCM replicative helicase motor are sequentially loaded onto replication origins by the origin recognition complex (ORC) as a head-to-head double hexamer complex. Although extensively studied in budding yeast, the mechanisms of origin licensing in multicellular eukaryotes remain poorly defined. The goal of my dissertation is to fill knowledge gaps in our understanding of metazoan origin licensing, with a focus on understanding how the MCM helicase is loaded in the absence of the defined initiator sequences that are used in yeast and the role of ORC's ORC6 subunit during this process. To address these questions, we report the use of biochemical reconstitution and electron microscopy (EM) to reconstruct the human MCM loading pathway. I find that, unlike in yeast, the ORC6 subunit is not essential for but enhances human MCM loading efficiency. EM analyses identify several intermediates en route to MCM double hexamer formation in the presence and absence of ORC6, including a DNA-loaded, closed-ring MCM single hexamer intermediate that can mature into a head-to-head double hexamer through multiple mechanisms that are distinct from the yeast pathway, including an ORC6-mediated pathway and two ORC6-independent pathways. I further investigate the evolutionary conservation of each of these pathways in the metazoan kingdom through an AlphaFold-guided phylogenetic analysis of metazoan origin licensing machinery, which reveals that the ORC6 protein has been lost in multiple, diverse metazoan lineages. Despite this loss, AlphaFold2 Multimer predictions suggests that an element in ORC3 which facilitates an ORC6-independent MCM loading mechanism is broadly conserved across the metazoan kingdom. Overall, my results suggest surprising heterogeneity in the methods by which metazoan species load the replicative helicase. This flexibility in MCM loading may provide resilience against cellular replication stress and a selective advantage for multicellular eukaryotes undergoing rapid cell division during early development, and the human reconstitution system paves the way to address outstanding questions regarding DNA replication initiation.

This document is currently not available here.

Share

COinS