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Redox Signaling in Eukaryotic DNA Replication and Repair

Citation

O'Brien, Elizabeth (2018) Redox Signaling in Eukaryotic DNA Replication and Repair. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/KGCP-SD98. https://resolver.caltech.edu/CaltechTHESIS:06112018-200314076

Abstract

DNA-mediated charge transport chemistry (DNA CT) offers an intriguing regulatory mechanism in biology, as it is long-range, rapid, and sensitive to mismatches and perturbations to base stacking. DNA-processing enzymes in all three domains of life moreover have been shown to contain [4Fe4S] clusters, commonly redox cofactors. Bacterial [4Fe4S] repair proteins have been shown to signal one another using long-range DNA-mediated charge transport (DNA CT), facilitating the redistribution to damaged genomic DNA in cells. The role of metabolically expensive, [4Fe4S] cluster cofactors in eukaryotic systems, however, was less clear than in prokaryotes.

Here we examine the chemical role of the [4Fe4S] cluster in eukaryotic DNA primase and the human base excision repair glycosylase, MUTYH. The primase cluster functions as a redox switch regulating DNA binding and redox signaling activity in humans and yeast. Yeast moreover require the primase redox switch for viability. Human MUTYH, a bifunctional glycosylase which repairs oxidative DNA lesions, performs DNA-mediated redox signaling, similarly to the bacterial homologue MutY. The MUTYH mutation which destabilizes the [4Fe4S] cluster during redox signaling, C306W, promotes degradation and loss of activity, associated with hereditary colorectal cancer.

To assess the redox role of the human primase [4Fe4S] cluster, we perform anaerobic DNA electrochemistry on the [4Fe4S] domain of human primase (p58C), which independently binds DNA. On DNA-modified Au electrodes, we compare the redox activity of electrochemically oxidized and electrochemically reduced p58C. Oxidized [4Fe4S]3+ p58C is electrochemically active, and reduced [4Fe4S]2+ p58C state is redox-inert. This redox-driven switch is electrochemically reversible, and is mediated by a triad of conserved tyrosines between the DNA binding interface and [4Fe4S] cluster. Mutation of residues Y309, Y345, and Y347 to phenylalanine causes attenuation of redox switching on DNA. Single-atom mutations in the redox pathway moreover compromise initiation and truncation of primer synthesis but do not affect RNA polymerase activity. We find that primase truncation is gated by DNA CT in vitro; a single mismatch in the nascent primer abrogates truncation of primase products. As primase is tethered to DNA polymerase α, a putative [4Fe4S] enzyme to which primase hands off the RNA-primed template, we propose that DNA-mediated signaling between primase and polymerase α chemically regulates this handoff during the first steps of replication.

Eukaryotic primase must bind both DNA and nucleotide triphosphates (NTPs) in order to convert to active form. Using DNA electrochemistry we show that p58C, and full-length DNA primase, display a robust, semi-reversible NTP-dependent signal on DNA, centered near 150mV vs. NHE. This signal is dependent on the tyrosine redox pathway. The presence of reversible redox activity at a physiological potential when primase is bound to DNA and NTPs suggests that reversible redox switching from the [4Fe4S]2+ to the [4Fe4S]3+ state is important for the activity of primase during replication.

The cluster serves as a redox switch governing DNA binding in yeast primase, just as in human primase. Mutation of tyrosines 395 and 397 in yeast primase moreover, alters the same electron transfer chemistry as the mutation of their orthologues, Y345 and Y347, respectively, alters in human primase. Although these tyrosines are arranged differently in the yeast and human proteins, they perform the same reaction to affect the switch. The single-atom Y395F mutation causes some sensitivity to chemically induced oxidative stress in yeast, and single-residue mutation Y397L confers lethality in yeast cells. A constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases, regulates the affinity for RNA-primed DNA template, and is required for primase function in vivo.

We finally characterize a novel mutation in the [4Fe4S] human base excision repair protein, MUTYH, which destabilizes the cluster environment and has pathogenic consequences. The MUTYH C306W mutation alters one of the cysteines coordinating the cluster to tryptophan. This mutation moreover is associated with hereditary colorectal cancer and causes defective DNA binding and enzymatic activity. We perform DNA electrochemistry on WT MUTYH, as well as C306W and two cancer-associated mutants, Y197C and G396D, which have an unaltered cluster environment. MUTYH variants participate in redox signaling, but C306W is destabilized upon oxidation from the [4Fe4S]2+ to the [4Fe4S]3+ state during signaling on DNA, leading to degradation to a [3Fe4S]+ cluster and loss of DNA binding and activity. A [4Fe4S] human DNA repair enzyme performs redox signaling on DNA; dysregulation of this signaling activity is linked to tumorigenesis.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:DNA Charge Transport, DNA Replication, Redox Signaling, Bioinorganic Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Barton, Jacqueline K.
Group:Kavli Nanoscience Institute
Thesis Committee:
  • Gray, Harry B. (chair)
  • Rees, Douglas C.
  • Tirrell, David A.
  • Barton, Jacqueline K.
Defense Date:1 June 2018
Record Number:CaltechTHESIS:06112018-200314076
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:06112018-200314076
DOI:10.7907/KGCP-SD98
Related URLs:
URLURL TypeDescription
https://doi.org/10.1002/ijch.201600022DOIArticle adapted for ch. 1
https://doi.org/10.1016/bs.mie.2017.03.024DOIArticle adapted for ch. 1
https://doi.org/10.1126/science.aag1789DOIArticle adapted for ch. 2
ORCID:
AuthorORCID
O'Brien, Elizabeth0000-0003-2889-1688
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11071
Collection:CaltechTHESIS
Deposited By: Elizabeth O'Brien
Deposited On:12 Jun 2018 20:56
Last Modified:18 Jun 2020 16:38

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