Holliday Junction Resolution Is Modulated by Archaeal Chromatin Components in Vitro

M. Kvaratskhelia, B.N. Wardleworth, Charlie Bond, J.M. Fogg, D.M.J. Lilley, M.F. White

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The Holliday junction-resolving enzyme Me is conserved in the archaea and probably plays a role analogous to that of Escherichia coli RuvC in the pathway of homologous recombination. Me specifically recognizes four-way DNA junctions, cleaving them without sequence preference to generate recombinant DNA duplex products. Hjc imposes an X-shaped global conformation on the bound DNA junction and distorts base stacking around the point of cleavage, three nucleotides 3' of the junction center. We show that Me is autoinhibitory under single turnover assay conditions and that this can be relieved by the addition of either competitor duplex DNA or the architectural double-stranded DNA-binding protein Sso7d (i.e. by approximating in vivo conditions more closely). Using a combination of isothermal titration calorimetry and fluorescent resonance energy transfer, we demonstrate that multiple Hjc dimers can bind to each synthetic four-way junction and provide evidence for significant distortion of the junction structure at high protein:DNA ratios. Analysis of crystal packing interactions in the crystal structure of Hjc suggests a molecular basis for this autoinhibition. The wider implications of these findings for the quantitative study of DNA-protein interactions is discussed.
Original languageEnglish
Pages (from-to)2992-2996
JournalJournal of Biological Chemistry
Volume277
Issue number4
DOIs
Publication statusPublished - 2002

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