Graduation Date

Summer 8-19-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Dr. Youri I. Pavlov

Abstract

During eukaryotic replication primase•polymerase α (prim•polα) complex synthesizes de novo chimeric primers composed of about 10 nt RNA and 20 nt DNA, which are subsequently extended by main replicative DNA polymerases (pol), polε and polδ, on leading and lagging strands, respectively. It is estimated that prim•polα initiates more than 10 millions of lagging strand Okazaki fragments in human genome in each replication cycle. A concerted action of the two active sites, RNA pol and DNA pol, is required to ensure the efficient priming. A remarkable feature of the prim•polα complex is the “programmed” synthesis of the chimeric primer, where the lengths of the RNA and DNA parts are tightly regulated. It is likely achieved by emerging intrinsic structural features of the complex and components of replication fork. To get a better understanding of the mechanism and biological importance of priming by the primase•polα, we utilized biochemical and genetic approaches to examine the protein-protein interactions, de novo synthesis and primer extension by prim•polα and the genome stability in primase mutants.

A direct interaction between the N-terminal domain of the human primase accessory subunit (p58N) and the C-terminal domain of the polα (p180C) catalytic subunit was found. The function of the C-terminal domain of primase containing Fe-S cluster and linker connecting it with p58N in the regulation of primase and polα synthesis was revealed. A novel interaction between the C-terminal domain with the 5’ triphosphate group of the RNA primer as well as the phosphodiester backbone of the template at the primer/template junction defines the length of the RNA primer, and the position where polα synthesis starts. We describe two mechanisms of decrease of apparent processivity of polα induced by divalent metal ions. The increased genomic instability in yeast mutants defective in primer initiation during Okazaki fragment synthesis led to a hypothesis on mutation-prone Okazaki fragment maturation when priming is delayed.

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