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ORCID

http://orcid.org/0000-0003-2713-5602

Date of Award

Summer 8-15-2020

Author's School

Graduate School of Arts and Sciences

Author's Department

Physics

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The homo-tetrameric E. coli single strand (ss) DNA binding (SSB) protein is an essential component in DNA maintenance for its role in binding and protecting single stranded DNA intermediates via its N-terminal DNA binding domain (DBD). SSB also acts as a hub to recruit at least 17 SSB interacting proteins (SIPs) involved in DNA replication, recombination, and repair via its 9 amino acid C-terminal acidic tip region. A 56 amino acid intrinsically disordered linker connects the DBD and the acidic tip and plays a role in cooperative binding to ssDNA. Using isothermal titration calorimetry, I determined that the SSB-Ct peptides bind to different SIPs with specificity, with affinity decreasing in the order: RecO > PriA ~ c subunit of DNA Pol III > PriC. I also determined that the intrinsically disordered linker itself does not contribute to SIP binding. There are, however, additional interactions between the DBD of SSB and RecO. Furthermore, as the acidic tip region can interact intramolecularly with the DBD of SSB, RecO is in competition with the DBD to bind to the acidic tip. SSB-Ct binding to DBD is eliminated when the DBD is occupied by ssDNA.

E. coli RecO is a recombination mediator protein (RMP) that is essential in one of the two major pathways of DNA repair, and interacts with an accessory protein, RecR. I investigated the oligomeric assembly properties of E. coli RecO and RecR proteins as well as the RecOR complexes using analytical ultracentrifugation, both by sedimentation velocity and sedimentation equilibrium. I found that E. coli RecR exists in a pH-dependent dimer-tetramer equilibrium, and that the dimeric state is stabilized at higher pH. However, I found that monomeric RecO forms complexes with only the RecR tetramer forming two RecOR complex species, RecR4O and RecR4O2. Investigating the DNA binding activity of RecO, I observed with light scattering measurements and confocal microscopy imaging that RecO-DNA complexes form aggregates with the propensity to form aggregates increasing with the length of DNA. Binding of RecO to either an SSB-Ct containing peptide or RecR inhibits aggregate formation.

The direct interactions between SSB and SIPs have been shown to influence various activities of the SIPs. I observed multiple allosteric effects of SSB-Ct peptides on RecOR complex formation and RecO(R) binding to ssDNA. While E. coli RecR does not interact with SSB or DNA, I found that SSB-Ct peptides stabilize the RecR4O complex over R4O2. Furthermore, SSB-Ct peptides allosterically enhance binding of RecO to ssDNA but reduce binding of RecOR complex to ssDNA. From these observations, I suggest a mechanism by which SSB recruits RecO and RecR proteins to a damaged DNA site facilitating the loading of RecA protein to initiate homologous recombination.

Language

English (en)

Chair and Committee

Anders E. Carlsson Timothy Lohman

Committee Members

Roberto Galletto, Shankar Mukherji, Mikhail Tikhonov,

Available for download on Sunday, July 24, 2022

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