Visualizing DNA polymerase ι catalyze Hoogsteen-directed DNA synthesis

Zach Frevert; Devin T. Reusch; Melissa S. Gildenberg; Sarah M. Jordan; Benjamin J. Ryan; Bret D. Freudenthal; M. Todd Washington

doi:10.1038/s41467-025-61245-8

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Visualizing DNA polymerase ι catalyze Hoogsteen-directed DNA synthesis

Journal article

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Visualizing DNA polymerase ι catalyze Hoogsteen-directed DNA synthesis

Zach Frevert, Devin T. Reusch, Melissa S. Gildenberg, Sarah M. Jordan, Benjamin J. Ryan, Bret D. Freudenthal and M. Todd Washington

Nature communications, Vol.16(1), 5979

07/01/2025

DOI: 10.1038/s41467-025-61245-8

PMCID: PMC12214486

PMID: 40593703

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Abstract

Translesion synthesis polymerases efficiently incorporate nucleotides opposite DNA lesions. Pol ι, for example, bypasses minor-groove and exocyclic purine adducts. Conventional X-ray crystallography showed that this enzyme incorporates nucleotides by forming Hoogsteen base pairs with the incoming nucleotide rather than Watson-Crick base pairs. While this revealed the structural basis of nucleotide selection during nucleotide binding, it did not allow the visualization of the process of phosphodiester bond formation or the detection of reaction intermediates that form during nucleotide incorporation. Here, we use a combination of time-lapse crystallography and molecular dynamics simulations to examine the mechanism of pol ι-catalyzed nucleotide incorporation. We show that this enzyme maintains Hoogsteen base pairing with the incoming dNTP during the entire reaction. We also show that pol ι possesses a pyrophosphatase activity that generates two monophosphates within its active site. Our findings provide insights into the features of pol ι’s active site that allow it to translocate along DNA and catalyze processive DNA synthesis. Pol ι forms Hoogsteen base pairs with the incoming nucleotide. Here, the authors use time-lapse X-ray crystallography to show that Hoogsten base pairing is maintained within the pol ι active site throughout the nucleotide incorporation reaction.

Details

Title: Subtitle: Visualizing DNA polymerase ι catalyze Hoogsteen-directed DNA synthesis
Creators: Zach Frevert - University of Iowa
Devin T. Reusch - University of Iowa
Melissa S. Gildenberg - University of Iowa
Sarah M. Jordan - Iowa City, IA USA
Benjamin J. Ryan - University of Kansas Medical Center
Bret D. Freudenthal - University of Kansas Medical Center
M. Todd Washington - University of Iowa
Resource Type: Journal article
Publication Details: Nature communications, Vol.16(1), 5979
DOI: 10.1038/s41467-025-61245-8
PMID: 40593703
PMCID: PMC12214486
NLM abbreviation: Nat Commun
ISSN: 2041-1723
eISSN: 2041-1723
Publisher: Nature Publishing Group UK
Grant note: U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS): R35 GM148186, R35-GM128562 Nation Institute of General Medical Sciences
This work was supported by award R35 GM148186 to M.T.W. and by award R35-GM128562 to B.D.F. from the Nation Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institute of General Medical Sciences or the National Institutes of Health. We thank Lynne Dieckman, Christine Kondratick, Justin Ling, and Tyler Woodward for discussions. We thank Michael Schnieders and Adrian Elcock for their assistance in setting up the simulations.
Language: English
Date published: 07/01/2025
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984843746302771

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