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Date of Award

Summer 8-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The work described in this dissertation focuses on several aspects of DNA replication in the model organism Saccharomyces cerevisiae, with particular attention paid to the function of the replicative DNA polymerase delta (Pol ), and its functions in Okazaki fragment synthesis and maturation. The first major theme of this dissertation is investigating the role that metal binding motifs play in the structure and function of Pol and other budding yeast polymerases. First, I discuss the role that two metal binding motifs within the catalytic subunit of Pol play in creating the multi-subunit polymerase complex and in promoting crucial interactions with the replication sliding clamp, proliferating cell nuclear antigen (PCNA). Next, I describe work defining the importance of similar metal binding motifs in the translesion DNA polymerase (Pol ). This yielded the observation that the two accessory subunits of Pol, Pol31 and Pol32, are also constitutive members of a four-subunit Pol complex. Finally, I describe the creation of a chimeric DNA polymerase comprising the bacteriophage RB69 DNA polymerase fused to the metal binding domain of the Pol catalytic subunit. We show that this chimeric polymerase can form a multimeric complex containing the Pol accessory subunits, interact with PCNA, and support DNA replication in vivo. This data provided insight into the structural requirements of the lagging strand replication machinery.

The second major theme is Pol s crucial role in synthesizing Okazaki fragments and participating in the removal of initiator RNA, called Okazaki fragment maturation. I first describe my work developing a system to study the activity of Pol in higher kinetic detail than previous studies, using rapid-quench techniques. This work yielded insights into how Pol performs DNA synthesis and strand displacement synthesis, as well as accomplishes nick translation, requiring collaboration between Pol and the flap-endonuclease FEN1. The next chapter describes the production and characterization of engineered PCNA heterotrimers. These proteins were produced to test the toolbelt model, which is the hypothesis that PCNA binds multiple enzymes simultaneously to increase the efficiency of DNA metabolism processes involving multiple enzymes. Finally, there has been a growing interest among those studying lagging strand synthesis into how potential impediments to the Okazaki fragment maturation machinery are resolved; we show that although the transcription factor Rap1 can block strand displacement synthesis by Pol when it is bound to DNA, this block can be resolved through the action of the helicase Pif1. In sum, these studies provide insight into how Pol s structure dictates its function, as well as addresses larger mechanistic questions concerning how lagging strand DNA replication is accomplished.

Language

English (en)

Chair and Committee

Peter Burgers

Committee Members

Roberto Galletto, Timothy Lohman, Sheila Stewart, Heather True

Comments

Permanent URL: https://doi.org/10.7936/K7GX48VX

Available for download on Saturday, August 15, 2116

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