Book of Abstracts: Albany 2005
Characterization of HU-cruciform DNA Interaction
Fundamental cellular processes such as DNA packaging, replication, recombination, and transcription (1, 2) are mediated by higher-order protein-DNA complexes. Their assembly involves the participation and cooperation between numerous proteins and DNA sites. Important factors in nucleoprotein complex formation are local DNA flexibility, a reflection of its physical properties, and DNA flexers, proteins that bind DNA and upon binding facilitate different DNA distortions (curvature, loop, hairpin, wrapping).
The Escherichia coli HU protein is regarded as a DNA flexer and is implicated in several cellular processes in which the HU-DNA interaction has the same outcome: higher-order nucleoprotein complex formation. HU is a non-sequence specific DNA binding protein, where binding is largely mediated through electrostatic interactions. In addition to non-specific binding, HU has been shown to bind with higher affinity to discontinuous duplex DNA containing structural modifications such as a nick or gap of one or two nucleotides and DNA cruciforms (3, 4). Cruciform DNA adopts a relaxed or stacked conformation depending on the presence or absence of salt.
In this work a structural and energetic analysis of the structurally specific HU-cruciform DNA interaction using spectroscopic methods is performed. Fluorescence intensity measurements have been used to characterize binding interactions. Anisotropy and fluorescence intensity measurements of HU binding to rhodamine and fluoresceine 5'labeled cruciform DNAs yield comparable binding affinities as those observed in gel studies (Kd = 6nM-10nM in 200mM NaCl and 30nM in 100 µM MgCl2). FRET measurements performed under different ionic strengths (0-1mM) and in the presence of different monovalent (K+, Na+) and divalent counterions (Mg2+) confirmed that the cruciform DNA adopts a stacked ?X? conformation at higher ion concentration. Structural changes induced by HU binding are monitored and compared with the ion-induced changes on the same cruciform substrate. Our experiments indicate that HU binding induces a greater stacked conformation relative to the ion-induced conformation.
Reference and Footnotes