Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

Lalith Perera, Bret D. Freudenthal, William A. Beard, David D. Shock, Lee G. Pedersen, Samuel H. Wilson, Suse Broyde

Research output: Contribution to journalArticle

Abstract

DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3′ of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase β active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophos-phorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.

Original languageEnglish (US)
Pages (from-to)E5228-E5236
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number38
DOIs
StatePublished - Sep 22 2015

Fingerprint

DNA-Directed DNA Polymerase
Metals
Ions
Catalytic Domain
Nucleotides
Magnesium
Genomic Instability
Site-Directed Mutagenesis
DNA Replication
DNA Repair
Binding Sites

Keywords

  • DNA polymerase
  • DNA repair
  • Pyrophosphorolysis
  • QM/MM
  • Reaction mechanism

ASJC Scopus subject areas

  • General

Cite this

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse. / Perera, Lalith; Freudenthal, Bret D.; Beard, William A.; Shock, David D.; Pedersen, Lee G.; Wilson, Samuel H.; Broyde, Suse.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, No. 38, 22.09.2015, p. E5228-E5236.

Research output: Contribution to journalArticle

Perera, Lalith ; Freudenthal, Bret D. ; Beard, William A. ; Shock, David D. ; Pedersen, Lee G. ; Wilson, Samuel H. ; Broyde, Suse. / Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse. In: Proceedings of the National Academy of Sciences of the United States of America. 2015 ; Vol. 112, No. 38. pp. E5228-E5236.
@article{407ebe0093044526965565e0afed12dc,
title = "Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse",
abstract = "DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is {"}balanced,{"} as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3′ of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase β active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophos-phorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.",
keywords = "DNA polymerase, DNA repair, Pyrophosphorolysis, QM/MM, Reaction mechanism",
author = "Lalith Perera and Freudenthal, {Bret D.} and Beard, {William A.} and Shock, {David D.} and Pedersen, {Lee G.} and Wilson, {Samuel H.} and Suse Broyde",
year = "2015",
month = "9",
day = "22",
doi = "10.1073/pnas.1511207112",
language = "English (US)",
volume = "112",
pages = "E5228--E5236",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "38",

}

TY - JOUR

T1 - Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

AU - Perera, Lalith

AU - Freudenthal, Bret D.

AU - Beard, William A.

AU - Shock, David D.

AU - Pedersen, Lee G.

AU - Wilson, Samuel H.

AU - Broyde, Suse

PY - 2015/9/22

Y1 - 2015/9/22

N2 - DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3′ of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase β active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophos-phorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.

AB - DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3′ of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase β active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophos-phorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.

KW - DNA polymerase

KW - DNA repair

KW - Pyrophosphorolysis

KW - QM/MM

KW - Reaction mechanism

UR - http://www.scopus.com/inward/record.url?scp=84942904891&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84942904891&partnerID=8YFLogxK

U2 - 10.1073/pnas.1511207112

DO - 10.1073/pnas.1511207112

M3 - Article

VL - 112

SP - E5228-E5236

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 38

ER -