| Summary: | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010. === Cataloged from PDF version of thesis. === Includes bibliographical references. === Eukaryotic chromosomal DNA replication is a tightly regulated process that initiates at multiple origins of replication throughout the genome. As cells enter the GI phase of the cell cycle, the Mcm2-7 replicative helicase is loaded at all potential origins of replication but remains inactive until S phase. Although the proteins involved in helicase loading have been elucidated, little is understood about the role of origin DNA sequence in helicase loading beyond its role in recruiting the initiator ORC. Of the eukaryotic origins of replication studied, those derived from the budding yeast Saccharomyces cerevisiae are most defined. These origins contain multiple functional DNA elements: a conserved and essential autonomously replicating sequence (ARS) consensus sequence (ACS) and multiple non-conserved B-elements that collectively are essential for origin function. Although the ACS and one of the B elements (B 1) have been demonstrated to bind ORC, the functions of the other Belements are poorly understood. In vivo studies indicate that the B2-element functions during helicase loading, but whether B2 is important for the initial recruitment or for the stable loading of the helicase (i.e. topological linkage around DNA) had not been determined. To understand how origin DNA facilitates helicase loading, I identified the specific DNA length and sequence requirements for helicase loading. I found that Mcm2-7 helicases are predominantly located at the region containing the B-elements, termed the B-side, at origins in vivo. This region coincides with a nucleosome-free region associated with each origin. Deletion of sequences within this region had no effect on the initial association of Mcm2-7 complexes to the origin, but either reduced or eliminated stable Mcm2-7 loading. In vitro assays that restricted the accessibility of ORC-adjacent DNA, either by the presence of nucleosomes or by truncation, revealed a function for B2 during helicase loading. Further analysis showed that B2 functions after the initial association of Mcm2-7 to facilitate helicase loading. This work provides insights into how origin sequences facilitate the specific protein/DNA and nucleosomal architecture necessary for helicase loading at eukaryotic origins of replication. === by Wendy M. Lam. === Ph.D.
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