Structure of the tertiary complex of the RepA hexameric helicase of plasmid RSF1010 with the ssDNA and nucleotide cofactors in solution

Agnieszka Marcinowicz, Maria J. Jezewska, Pawel Bujalowski, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

The structure of the complex of the hexameric replicative helicase RepA protein of plasmid RSF1010 with ssDNA has been examined using the fluorescence energy transfer and analytical ultracentrifugation methods. We utilized the fact that the RepA monomer contains a single, natural cysteine residue. The cysteine residue has been modified with a fluorescent marker, which serves as the donor to the acceptor placed in different locations on the DNA. Using the two independent fluorescence donor-acceptor pairs and different DNA oligomers, we provide direct evidence that, in the complex with the enzyme, the ssDNA passes through the inner channel of the RepA hexamer. In the stationary complex, the RepA hexamer assumes a strictly single orientation with respect to the polarity of the sugar-phosphate backbone of the nucleic acid, with the large domain of protomers facing the 3′ end of the bound DNA. Interactions with the helicase induce profound changes in the structure of the bound DNA, and these changes are predominantly localized in the proper DNA-binding site. The heterogeneity of the structure of the bound DNA reflects the heterogeneous structure of the total RepA helicase DNA-binding site. This is in excellent agreement with the thermodynamic data. The structure of the RepA hexamer, in solution, differs considerably from the crystal structure of the enzyme. Both fluorescence energy transfer and analytical ultracentrifugation data indicate a significant conformational flexibility of the RepA hexamer. Implications of these results for the mechanism of interactions of the hexameric helicase with the DNA are discussed.

Original languageEnglish (US)
Pages (from-to)13279-13296
Number of pages18
JournalBiochemistry
Volume46
Issue number46
DOIs
StatePublished - Nov 20 2007

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry

Cite this