The roles of the helicase double-hexamer complex and the ssDNA-binding protein RPA during eukaryotic DNA replication

Abstract

Eukaryotic DNA replication is a complex process that must occur accurately, completely, and only once per cell cycle. To accomplish these goals, the events of DNA replication are tightly coupled to cell-cycle progression. Origins of replication are licensed by loading of the Mcm2-7 replicative DNA helicase during G1. Two Mcm2-7 hexamers load onto each origin as a double hexamer encircling dsDNA. At this stage, the helicases are inactive. Upon entry into S phase, loaded Mcm2-7 complexes then recruit a number of other replication proteins that activate the helicase. Helicase activation results in separation of the double hexamer, a transition to encircling ssDNA, and initiation of DNA unwinding. Once activated, the helicase produces the ssDNA that acts as template for new DNA synthesis. Helicase activation is the committed step of DNA replication after which the cell must complete genome duplication before it can segregate its chromosomes and divide.

The work described in this thesis focuses on mechanisms that are essential for eukaryotic DNA replication with a focus on DNA unwinding and DNA synthesis. In Chapter II, I explore the essential functions and purpose of the double-hexamer conformation of the loaded helicases. Using a helicase mutant that loads as two single hexamers, I show that initial origin DNA melting can occur in the context of a single-hexamer helicase. Importantly, the amount of unwinding that occurs within a single helicase is not sufficient to allow the transition onto ssDNA. Further DNA unwinding and subsequent DNA synthesis requires robust double-hexamer helicase interactions. Together, my findings strongly suggest that the double-hexamer conformation is essential to complete helicase activation. In Chapter III, I explore the role and specificity of ssDNA-binding proteins (SSBs) in eukaryotic DNA replication. To this end, I substituted the eukaryotic SSB RPA with SSBs from other systems: E.

coli SSB (EcSSB) and T4 bacteriophage Gp32. I find that DNA unwinding is supported by RPA and EcSSB but not Gp32, suggesting that eukaryotic DNA unwinding requires at least one SSB function beyond ssDNA binding. Although both RPA and EcSSB support DNA synthesis, we only observed robust lagging-strand synthesis in the presence of RPA. My studies indicate that RPA must perform multiple functions beyond ssDNA binding to facilitate eukaryotic DNA replication.