Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV

Lindsey DeCarlo, A. S.Prakasha Gowda, Zucai Suo, Thomas Spratt

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol η. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce kpol and KddNTP values. A decrease in kpol/K ddNTP associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a > 1000-fold decrease in kpol/KddNTP, indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the kpol/KddNTP values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased > 10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.

Original languageEnglish (US)
Pages (from-to)8157-8164
Number of pages8
JournalBiochemistry
Volume47
Issue number31
DOIs
StatePublished - Aug 5 2008

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Sulfolobus solfataricus
DNA Polymerase beta
Base Pairing
Conformations
DNA
Hydrogen
Hydrogen bonds
Nucleotides
Guanine
Adenine
Substitution reactions
Dideoxynucleotides
DNA-Directed DNA Polymerase
DNA Damage
purine
Rate constants
Catalytic Domain
Kinetics
Geometry

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

DeCarlo, Lindsey ; Gowda, A. S.Prakasha ; Suo, Zucai ; Spratt, Thomas. / Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV. In: Biochemistry. 2008 ; Vol. 47, No. 31. pp. 8157-8164.
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abstract = "DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol η. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce kpol and KddNTP values. A decrease in kpol/K ddNTP associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a > 1000-fold decrease in kpol/KddNTP, indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the kpol/KddNTP values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased > 10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.",
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Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV. / DeCarlo, Lindsey; Gowda, A. S.Prakasha; Suo, Zucai; Spratt, Thomas.

In: Biochemistry, Vol. 47, No. 31, 05.08.2008, p. 8157-8164.

Research output: Contribution to journalArticle

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T1 - Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV

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AB - DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol η. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce kpol and KddNTP values. A decrease in kpol/K ddNTP associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a > 1000-fold decrease in kpol/KddNTP, indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the kpol/KddNTP values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased > 10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.

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