Kinetic mechanisms of rat polymerase β-ssNDA interactions. Quantitative fluorescence stopped-flow analysis of the formation of the (pol β)16 and (pol β)5 ssDNA binding mode

Maria J. Jezewska, Surendran Rajendran, Roberto Galletto, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Kinetics of rat polymerase β (pol β) binding to the single-stranded DNA (ssDNA) in the (pol β)16 and (pol β)5 binding modes has been examined, using the fluorescence stopped-flow technique. Binding of the enzyme to the ssDNA containing fluorescein is characterized by a strong increase of the DNA fluorescence, which provides an excellent signal to quantitatively study the complex mechanism of the ssDNA recognition process. The experiments were performed with a 20-mer ssDNA, which can engage the enzyme in the (pol β)16 binding mode, i.e. it encompasses the entire, total DNA-binding site of rat pol β, and with a 10-mer which binds the enzyme exclusively in the (pol β)5 binding mode where only the 8 kDa domain of the enzyme is engaged in interactions with the DNA. The data indicate that the formation of the (pol β)16 binding mode occurs by a minimum three-step mechanism with the bimolecular binding step followed by two isomerizations: A similar mechanism is observed in the formation of the (pol β)5 binding mode, although at low salt concentrations there is an additional, slow step in the reaction. The data analysis was performed using the matrix projection operator technique, a powerful method to address stopped-flow kinetics, particularly, amplitudes. The binding modes differ in the free energy changes of the partial reactions and ion effects on transitions between intermediates that reflect different participation of the two structural domains. The formation of both binding modes is initiated by the fast association with the ssDNA through the 8 kDa domain, followed by transitions induced by interactions at the interface of the 8 kDa domain and the DNA. In the (pol β)16 binding mode, the subsequent intermediates are stabilized by the DNA binding to the DNA-binding subsite on the 31 kDa domain. The data indicate that interactions of the ssDNA-binding subsite of the 8 kDa domain with the ssDNA, controlled by the ion binding, induce conformational transitions of the formed complexes in both binding modes. The sequential nature of the determined mechanisms indicates a lack of kinetically significant conformational equilibrium of rat pol β, prior to ssDNA binding.

Original languageEnglish (US)
Pages (from-to)977-1002
Number of pages26
JournalJournal of Molecular Biology
Volume313
Issue number5
DOIs
StatePublished - Nov 9 2001

Fingerprint

Single-Stranded DNA
Fluorescence
DNA
Enzymes
Ions
DNA-Directed DNA Polymerase
Fluorescein
Salts
Binding Sites

Keywords

  • Binding modes
  • DNA repair
  • Polymerases
  • Protein-DNA interactions
  • Stopped-flow kinetics

ASJC Scopus subject areas

  • Virology

Cite this

Kinetic mechanisms of rat polymerase β-ssNDA interactions. Quantitative fluorescence stopped-flow analysis of the formation of the (pol β)16 and (pol β)5 ssDNA binding mode. / Jezewska, Maria J.; Rajendran, Surendran; Galletto, Roberto; Bujalowski, Wlodzimierz.

In: Journal of Molecular Biology, Vol. 313, No. 5, 09.11.2001, p. 977-1002.

Research output: Contribution to journalArticle

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abstract = "Kinetics of rat polymerase β (pol β) binding to the single-stranded DNA (ssDNA) in the (pol β)16 and (pol β)5 binding modes has been examined, using the fluorescence stopped-flow technique. Binding of the enzyme to the ssDNA containing fluorescein is characterized by a strong increase of the DNA fluorescence, which provides an excellent signal to quantitatively study the complex mechanism of the ssDNA recognition process. The experiments were performed with a 20-mer ssDNA, which can engage the enzyme in the (pol β)16 binding mode, i.e. it encompasses the entire, total DNA-binding site of rat pol β, and with a 10-mer which binds the enzyme exclusively in the (pol β)5 binding mode where only the 8 kDa domain of the enzyme is engaged in interactions with the DNA. The data indicate that the formation of the (pol β)16 binding mode occurs by a minimum three-step mechanism with the bimolecular binding step followed by two isomerizations: A similar mechanism is observed in the formation of the (pol β)5 binding mode, although at low salt concentrations there is an additional, slow step in the reaction. The data analysis was performed using the matrix projection operator technique, a powerful method to address stopped-flow kinetics, particularly, amplitudes. The binding modes differ in the free energy changes of the partial reactions and ion effects on transitions between intermediates that reflect different participation of the two structural domains. The formation of both binding modes is initiated by the fast association with the ssDNA through the 8 kDa domain, followed by transitions induced by interactions at the interface of the 8 kDa domain and the DNA. In the (pol β)16 binding mode, the subsequent intermediates are stabilized by the DNA binding to the DNA-binding subsite on the 31 kDa domain. The data indicate that interactions of the ssDNA-binding subsite of the 8 kDa domain with the ssDNA, controlled by the ion binding, induce conformational transitions of the formed complexes in both binding modes. The sequential nature of the determined mechanisms indicates a lack of kinetically significant conformational equilibrium of rat pol β, prior to ssDNA binding.",
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N2 - Kinetics of rat polymerase β (pol β) binding to the single-stranded DNA (ssDNA) in the (pol β)16 and (pol β)5 binding modes has been examined, using the fluorescence stopped-flow technique. Binding of the enzyme to the ssDNA containing fluorescein is characterized by a strong increase of the DNA fluorescence, which provides an excellent signal to quantitatively study the complex mechanism of the ssDNA recognition process. The experiments were performed with a 20-mer ssDNA, which can engage the enzyme in the (pol β)16 binding mode, i.e. it encompasses the entire, total DNA-binding site of rat pol β, and with a 10-mer which binds the enzyme exclusively in the (pol β)5 binding mode where only the 8 kDa domain of the enzyme is engaged in interactions with the DNA. The data indicate that the formation of the (pol β)16 binding mode occurs by a minimum three-step mechanism with the bimolecular binding step followed by two isomerizations: A similar mechanism is observed in the formation of the (pol β)5 binding mode, although at low salt concentrations there is an additional, slow step in the reaction. The data analysis was performed using the matrix projection operator technique, a powerful method to address stopped-flow kinetics, particularly, amplitudes. The binding modes differ in the free energy changes of the partial reactions and ion effects on transitions between intermediates that reflect different participation of the two structural domains. The formation of both binding modes is initiated by the fast association with the ssDNA through the 8 kDa domain, followed by transitions induced by interactions at the interface of the 8 kDa domain and the DNA. In the (pol β)16 binding mode, the subsequent intermediates are stabilized by the DNA binding to the DNA-binding subsite on the 31 kDa domain. The data indicate that interactions of the ssDNA-binding subsite of the 8 kDa domain with the ssDNA, controlled by the ion binding, induce conformational transitions of the formed complexes in both binding modes. The sequential nature of the determined mechanisms indicates a lack of kinetically significant conformational equilibrium of rat pol β, prior to ssDNA binding.

AB - Kinetics of rat polymerase β (pol β) binding to the single-stranded DNA (ssDNA) in the (pol β)16 and (pol β)5 binding modes has been examined, using the fluorescence stopped-flow technique. Binding of the enzyme to the ssDNA containing fluorescein is characterized by a strong increase of the DNA fluorescence, which provides an excellent signal to quantitatively study the complex mechanism of the ssDNA recognition process. The experiments were performed with a 20-mer ssDNA, which can engage the enzyme in the (pol β)16 binding mode, i.e. it encompasses the entire, total DNA-binding site of rat pol β, and with a 10-mer which binds the enzyme exclusively in the (pol β)5 binding mode where only the 8 kDa domain of the enzyme is engaged in interactions with the DNA. The data indicate that the formation of the (pol β)16 binding mode occurs by a minimum three-step mechanism with the bimolecular binding step followed by two isomerizations: A similar mechanism is observed in the formation of the (pol β)5 binding mode, although at low salt concentrations there is an additional, slow step in the reaction. The data analysis was performed using the matrix projection operator technique, a powerful method to address stopped-flow kinetics, particularly, amplitudes. The binding modes differ in the free energy changes of the partial reactions and ion effects on transitions between intermediates that reflect different participation of the two structural domains. The formation of both binding modes is initiated by the fast association with the ssDNA through the 8 kDa domain, followed by transitions induced by interactions at the interface of the 8 kDa domain and the DNA. In the (pol β)16 binding mode, the subsequent intermediates are stabilized by the DNA binding to the DNA-binding subsite on the 31 kDa domain. The data indicate that interactions of the ssDNA-binding subsite of the 8 kDa domain with the ssDNA, controlled by the ion binding, induce conformational transitions of the formed complexes in both binding modes. The sequential nature of the determined mechanisms indicates a lack of kinetically significant conformational equilibrium of rat pol β, prior to ssDNA binding.

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