Biochemical evidence for the requirement of Hoogsteen base pairing for replication by human DNA polymerase ι

Robert E. Johnson, Louise Prakash, Satya Prakash

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Because of the near geometric identity of Watson-Crick (W-C) G·C and A·T base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase ι (Polι), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaadenine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Polι is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Polι active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, mutational studies with Leu-62, which lies in close proximity to the templating residue in the Polι ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Polι.

Original languageEnglish (US)
Pages (from-to)10466-10471
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number30
DOIs
StatePublished - Jul 26 2005

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DNA-Directed DNA Polymerase
Base Pairing
Purines
Catalytic Domain
Nucleotides
Pyrimidine Nucleotides
Pyrimidines
Hydrogen
DNA

Keywords

  • 7-deaza purines
  • Syn and anti conformations
  • Translesion DNA synthesis

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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title = "Biochemical evidence for the requirement of Hoogsteen base pairing for replication by human DNA polymerase ι",
abstract = "Because of the near geometric identity of Watson-Crick (W-C) G·C and A·T base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase ι (Polι), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaadenine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Polι is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Polι active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, mutational studies with Leu-62, which lies in close proximity to the templating residue in the Polι ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Polι.",
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N2 - Because of the near geometric identity of Watson-Crick (W-C) G·C and A·T base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase ι (Polι), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaadenine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Polι is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Polι active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, mutational studies with Leu-62, which lies in close proximity to the templating residue in the Polι ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Polι.

AB - Because of the near geometric identity of Watson-Crick (W-C) G·C and A·T base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase ι (Polι), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaadenine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Polι is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Polι active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, mutational studies with Leu-62, which lies in close proximity to the templating residue in the Polι ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Polι.

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