Date of Award

Spring 2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Antony, Edwin P.

Second Advisor

Maurice, Martin St.

Third Advisor

Manogaran, Anita L.

Abstract

During DNA metabolic processes such as replication and repair, double-stranded DNA is transiently unwound to expose single-stranded DNA (ssDNA). Such ssDNA intermediates are immediately coated by Replication Protein A (RPA), an essential single-stranded DNA binding protein present in all eukaryotes. RPA binding to ssDNA fulfills four goals: 1. The ssDNA is protected from degradation by endo- and exo-nucleases. 2. A cell-cycle checkpoint signaling cascade is triggered to indicate the presence of ssDNA. 3. RPA recruits other DNA metabolic enzymes on to the ssDNA. 4. Finally, RPA promotes the catalytic activity of the recruited enzyme. The overall objective of my thesis work focuses on deciphering how a single enzyme can coordinate multiple DNA metabolic processes in the cell to protect genomic integrity.RPA is a multi-subunit complex composed of four DNA binding domains (DBDs-A, B, C and D) and two protein interaction domains (PIDs). The DBDs promote high affinity binding to ssDNA and the PIDs interact with more than two dozen proteins to control and coordinate various DNA metabolic processes. The DBDs and PIDs are tethered by flexible disordered linkers that enable RPA to adopt a variety of conformations. A long-standing hypothesis posits that specific RPA-conformations drive a corresponding cellular activity. My thesis work has developed experimental methodologies to visualize and capture the conformational states of RPA. Furthermore, we show how RPA-conformations are modulated by RPA-interacting proteins and post-translational modifications.The major findings of my thesis research are: i) Establishment of DBDs A, B as the flexible unit of RPA, and DBDs C, D with RPA14 as the stable unit of RPA. ii) Existence of four distinct microscopic binding/ dissociation states of DBD-A and DBD-D in context of full-length RPA iii) DNA structures encountered by RPA affect the conformational states and arrangement of RPA-DBDs iv) RPA interacting proteins, such as Rad52, can selectively modify the DNA binding dynamics of a particular DBD. v) Cooperative binding in Rad52 selectively modulates the DNA binding states of DBD-D but not DBA-A and vi) Post-translational modification alters microscopic binding states and conformational arrangement of DBDs on DNA. This alteration can affect both assembly and stability of the RPA-DNA complex alone, or in the presence of proteins seeking to displace RPA from DNA.

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