Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

Bret D. Freudenthal, William A. Beard, Lalith Perera, David D. Shock, Taejin Kim, Tamar Schlick, Samuel H. Wilson

Research output: Contribution to journalArticle

Abstract

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'2-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

Original languageEnglish (US)
Pages (from-to)635-639
Number of pages5
JournalNature
Volume517
Issue number7536
DOIs
StatePublished - Jan 29 2015

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Nucleotides
Cytosine
Adenine
Mutagenesis
Base Pairing
DNA Repair
Anti-Bacterial Agents
Crystallography
Deoxyguanosine
Genomic Instability
Guanine
DNA-Directed DNA Polymerase
Enzymes
Hydrogen Bonding
8-oxodeoxyguanosine triphosphate
Catalysis
Nucleosides
Neoplasms
Cell Survival
Oxidative Stress

ASJC Scopus subject areas

  • General

Cite this

Freudenthal, B. D., Beard, W. A., Perera, L., Shock, D. D., Kim, T., Schlick, T., & Wilson, S. H. (2015). Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. Nature, 517(7536), 635-639. https://doi.org/10.1038/nature13886

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. / Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; Shock, David D.; Kim, Taejin; Schlick, Tamar; Wilson, Samuel H.

In: Nature, Vol. 517, No. 7536, 29.01.2015, p. 635-639.

Research output: Contribution to journalArticle

Freudenthal, BD, Beard, WA, Perera, L, Shock, DD, Kim, T, Schlick, T & Wilson, SH 2015, 'Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide', Nature, vol. 517, no. 7536, pp. 635-639. https://doi.org/10.1038/nature13886
Freudenthal BD, Beard WA, Perera L, Shock DD, Kim T, Schlick T et al. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. Nature. 2015 Jan 29;517(7536):635-639. https://doi.org/10.1038/nature13886
Freudenthal, Bret D. ; Beard, William A. ; Perera, Lalith ; Shock, David D. ; Kim, Taejin ; Schlick, Tamar ; Wilson, Samuel H. / Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. In: Nature. 2015 ; Vol. 517, No. 7536. pp. 635-639.
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