Fidelity of mispair formation and mispair extension is dependent on the interaction between the minor groove of the primer terminus and Arg668 of DNA polymerase I of Escherichia coli

Melodie D. McCain, Aviva S. Meyer, Sherri S. Schultz, Athanasios Glekas, Thomas Spratt

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

The hydrogen bonding interactions between the Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease inactivated (KF-) and the minor groove of DNA were examined with modified oligodeoxynucleotides in which 3-deazaguanine (3DG) replaced guanine. This substitution would prevent a hydrogen bond from forming between the polymerase and that one site on the DNA. If the hydrogen bonding interaction were important, then we should observe a decrease in the rate of reaction. The steady-state and pre-steady-state kinetics of DNA replication were measured with 10 different oligodeoxynucleotide duplexes in which 3DG was placed at different positions. The largest decrease in the rate of replication was observed when 3DG replaced guanine at the 3′-terminus of the primer. The effect of this substitution on mispair extension and formation was then probed. The G to 3DG substitution at the primer terminus decreased the kpol for the extension past G/C, G/A, and G/G base pairs but not the G/T base pair. The G to 3DG substitution at the primer terminus also decreased the formation of correct base pairs as well as incorrect base pairs. However, in all but two mispairs, the effect on correct base pairs was much greater than that of mispairs. These results indicate that the hydrogen bond between Arg668 and the minor groove of the primer terminus is important in the fidelity of both formation and extension of mispairs. These experiments support a mechanism in which Arg668 forms a hydrogen bonding fork between the minor groove of the primer terminus and the ring oxygen of the deoxyribose moiety of the incoming dNTP to align the 3′-hydroxyl group with the α-phosphate of the dNTP. This is one mechanism by which the polymerase can use the geometry of the base pairs to modulate the rate of formation and extension of mispairs.

Original languageEnglish (US)
Pages (from-to)5647-5659
Number of pages13
JournalBiochemistry
Volume44
Issue number15
DOIs
StatePublished - Apr 19 2005

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DNA Polymerase I
Base Pairing
Escherichia coli
Hydrogen bonds
Substitution reactions
Hydrogen Bonding
Oligodeoxyribonucleotides
Guanine
DNA
Hydrogen
Deoxyribose
Exonucleases
Hydroxyl Radical
DNA Replication
Phosphates
3-deazaguanine
Oxygen
Kinetics
Geometry
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Fidelity of mispair formation and mispair extension is dependent on the interaction between the minor groove of the primer terminus and Arg668 of DNA polymerase I of Escherichia coli",
abstract = "The hydrogen bonding interactions between the Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease inactivated (KF-) and the minor groove of DNA were examined with modified oligodeoxynucleotides in which 3-deazaguanine (3DG) replaced guanine. This substitution would prevent a hydrogen bond from forming between the polymerase and that one site on the DNA. If the hydrogen bonding interaction were important, then we should observe a decrease in the rate of reaction. The steady-state and pre-steady-state kinetics of DNA replication were measured with 10 different oligodeoxynucleotide duplexes in which 3DG was placed at different positions. The largest decrease in the rate of replication was observed when 3DG replaced guanine at the 3′-terminus of the primer. The effect of this substitution on mispair extension and formation was then probed. The G to 3DG substitution at the primer terminus decreased the kpol for the extension past G/C, G/A, and G/G base pairs but not the G/T base pair. The G to 3DG substitution at the primer terminus also decreased the formation of correct base pairs as well as incorrect base pairs. However, in all but two mispairs, the effect on correct base pairs was much greater than that of mispairs. These results indicate that the hydrogen bond between Arg668 and the minor groove of the primer terminus is important in the fidelity of both formation and extension of mispairs. These experiments support a mechanism in which Arg668 forms a hydrogen bonding fork between the minor groove of the primer terminus and the ring oxygen of the deoxyribose moiety of the incoming dNTP to align the 3′-hydroxyl group with the α-phosphate of the dNTP. This is one mechanism by which the polymerase can use the geometry of the base pairs to modulate the rate of formation and extension of mispairs.",
author = "McCain, {Melodie D.} and Meyer, {Aviva S.} and Schultz, {Sherri S.} and Athanasios Glekas and Thomas Spratt",
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Fidelity of mispair formation and mispair extension is dependent on the interaction between the minor groove of the primer terminus and Arg668 of DNA polymerase I of Escherichia coli. / McCain, Melodie D.; Meyer, Aviva S.; Schultz, Sherri S.; Glekas, Athanasios; Spratt, Thomas.

In: Biochemistry, Vol. 44, No. 15, 19.04.2005, p. 5647-5659.

Research output: Contribution to journalArticle

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AU - McCain, Melodie D.

AU - Meyer, Aviva S.

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