Energetics and Specificity of Rat DNA Polymerase β Interactions with Template-primer and Gapped DNA Substrates

Maria J. Jezewska, Surendran Rajendran, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Interactions between rat polymerase β (pol β) and the template-primer, as well as gapped DNAs, were studied using the quantitative fluorescence titration technique. Stoichiometries of rat pol β complexes with DNA substrates are much higher than stoichiometries predicted by the structures of co-crystals. The data can be understood in the context of the two single-stranded (ss)DNA-binding modes of the enzyme, the (pol β) 16 and (pol β)5 binding modes, which differ by the number of nucleotides occluded by the protein. The 8-kDa domain of the enzyme engages the double-stranded (ds)DNA down-stream from the primer, while the 31-kDa domain has similar affinity for the ss-ds DNA junction and the dsDNA. The affinity of rat pol β for the gapped DNA is not affected by the size of the gap. The results indicate a plausible model for recognition of the gapped DNA by rat pol β. The enzyme binds the ss-ds DNA junction of the gap using the 31-kDa domain. This binding induces an allosteric transition, resulting in the association of the 8-kDa domain with the dsDNA, leading to an amplification of the affinity for the gap. The 5′ terminal phosphate, downstream from the primer, has little effect on the affinity, but affects the ssDNA conformation of the gap.

Original languageEnglish (US)
Pages (from-to)16123-16136
Number of pages14
JournalJournal of Biological Chemistry
Volume276
Issue number19
DOIs
StatePublished - May 11 2001

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DNA Primers
DNA-Directed DNA Polymerase
Rats
DNA
Substrates
Stoichiometry
Enzymes
Single-Stranded DNA
Gap Junctions
Titration
Amplification
Conformations
Nucleotides
Fluorescence
Phosphates
Association reactions
Crystals
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Energetics and Specificity of Rat DNA Polymerase β Interactions with Template-primer and Gapped DNA Substrates. / Jezewska, Maria J.; Rajendran, Surendran; Bujalowski, Wlodzimierz.

In: Journal of Biological Chemistry, Vol. 276, No. 19, 11.05.2001, p. 16123-16136.

Research output: Contribution to journalArticle

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