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Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways


Zhou, Andy (2019) Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/G7NF-S349.


The 4Fe4S cluster has been identified in various DNA-processing proteins spanning a variety of biological functions and all domains of life. Recently, a novel functional role for the cluster has been identified for proteins in DNA repair and replication as a redox switch for DNA binding. Human DNA primase utilizes this redox switch to coordinate primer handoff in replication. The enzymatic activity of DNA polymerase δ is tuned by the redox-switch, allowing for a fast and reversible regulation of replication in response to oxidative stress. In all cases, the redox of the 4Fe4S cluster is achieved through DNA-mediated charge transport (CT), the ability for DNA to carry charge through its π-stack. Due to the reliance of this phenomena on the π-stacking of the nitrogenous bases, DNA CT is sensitive to DNA lesions and mismatches and can proceed over long molecular distances if the DNA is well-stacked. Given this powerful biological phenomena, new inter-protein signaling interactions have been identified with important downstream consequences for genome fidelity. Here, we investigate the ways DNA-mediated charge transport between DNA processing enzymes results in efficient DNA repair or prevention of DNA-damage.

First, we investigated Dps, a bacterial ferritin that protects DNA from oxidative stress and implicated in bacterial survival and virulence. Dps iron sites can scavenge diffusing oxidants directly but additionally electrons and electron holes can be rapidly transported through the base-pair π-stack though DNA CT, thus providing an additional mechanism of genome protection by Dps. Using X-band EPR, we monitored formation of mononuclear high-spin Fe(III) sites of low symmetry as a gauge of effective Dps protection via oxidation of its iron sites. Using poly(dGdC)2 or poly(dAdT)2 DNA, we uncovered the dependence of DNA protection by Dps to the formation of guanine radical intermediates. Oxidation of Dps iron sites depended on the presence of the W52 residue. Point mutations of W52 revealed its involvement in an electron transfer (ET) pathway for the oxidation of the Dps iron sites. Finally, we investigated the in vivo consequences of the Dps W52 residue by complementing knockout Dps E.coli with plasmids expressing WT, W52A, or W52Y Dps and applying oxidative stress to the cells through hydrogen peroxide treatment. These assays further demonstrated the ability of Dps to protect the E.coli genome from harmful oxidants DNA-mediated electron transfer processes.

Second, we assessed the redox properties of EndoIII and MutY, two base excision repair glycosylases containing 4Fe4S clusters, in the presence and absence of DNA. Previous work has shown these proteins to have a midpoint redox potential around 80mV vs. NHE when bound to DNA with a positive shift in potential in the absence of DNA. However, electrochemical details that define this midpoint potential have not been uncovered. Using a pyrolytic graphite edge electrode, we measured the midopoint potential of point mutations of EndoIII where point charges are flipped near the cluster (K208E, Y205H, and E200K) in the absence of DNA. Our measurements suggest that a change in a single point charge is not enough to shift the 4Fe4S cluster midpoint potential dramatically. Addition of a poly-L-glutamate polyanion introduced a slight negative shift (~20mV), but with the introduction of DNA a large negative shift was observed (70mV). Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the 4Fe4S cluster, helping to explain why all DNA binding proteins with 4Fe4S clusters studied to date have similar DNA-bound potentials.

With these similar DNA-bound potentials, inter-protein redox signaling should occur. Previous works have demonstrated DNA-mediated redox signaling such as EndoIII signaling to DinG helicase, involved in R-loop maturation, increasing cellular survival by resolving deleterious R-loops. Additionally, different cluster- containing repair proteins of different functions and domains of life have been shown using atomic force microscopy (AFM) to localize to DNA mismatches through a redox switch for DNA-binding affinity. Given a DNA-mediated redox signaling system to scan the genome for lesions, the expression levels of these proteins may play a role in defining the scanning efficiency. We identified that the EndoIII E.coli knockout strain was sensitive to UV irradiation. This implies that EndoIII assists the nucleotide excision repair (NER) pathway via DNA-mediated redox signaling. However, knockout of MutY, another 4Fe4S glycosylase, does not impart the same UV sensitivity, and thus suggests key differences between MutY and EndoIII that define effective DNA-mediated redox signaling. Thus, the effect of protein expression level on the efficiency of DNA-mediated redox signaling was investigated using inducible protein expression of EndoIII to rescue UV-sensitivity. Using both plasmid-based and genome integrated constructs, we uncovered that low amounts of EndoIII expression were enough to rescue the growth defect, and overexpression of WT EndoIII leads to a greater defect caused by excess non-specific enzyme activity. These findings further informed investigation of this unique protein signaling interaction between EndoIII and NER protein UvrC.

With proper EndoIII rescue plasmids, we further characterized the DNA- mediated redox signaling interaction between EndoIII and UvrC. Using UV-irradiation of genetic knockout strains and growth curve analysis, we demonstrate that EndoIII expression is essential for efficient repair of UV-induced DNA lesions, as measured through quantitative changes in growth lag-time when wild-type or mutant EndoIII is present in the cell. Electrochemical analysis of EndoIII point mutants quantify the DNA-CT inefficiencies that lead to the observed phenotypes. EndoIII, a BER repair protein, assists the NER pathway in the repair of UV-induced DNA lesions via DNA-mediated redox signaling. These results give evidence of a new signaling crosstalk between two distinct DNA repair pathways.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:DNA repair; redox mediated signaling, DNA charge transport; Endonuclease III
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Barton, Jacqueline K.
Thesis Committee:
  • Gray, Harry B. (chair)
  • Rees, Douglas C.
  • Tirrell, David A.
  • Barton, Jacqueline K.
Defense Date:23 July 2018
Record Number:CaltechTHESIS:11262018-103442842
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Ch. 2. adapted for Ch. 3.
Zhou, Andy0000-0003-3383-0855
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11282
Deposited By: Andy Zhou
Deposited On:27 Nov 2018 19:32
Last Modified:08 Nov 2023 00:14

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