Flexibility In-Vitro, HMG Box Interactions, Saccharomyes-Cerevisiae, Crystal-Structure, Repression Loop, Faciliated Dissociation, Conformational-Changes, Single; HU, Complex
Journal/Book/Conference Title Title
Nucleic Acids Research
The yeast Nhp6A protein (yNhp6A) is a member of the eukaryotic HMGB family of chromatin factors that enhance apparent DNA flexibility. yNhp6A binds DNA nonspecifically with nM affinity, sharply bending DNA by >60◦. It is not known whether the protein binds to unbent DNA and then deforms it, or if bent DNA conformations are ‘captured’ by protein binding. The former mechanism would be supported by discovery of conditions where unbent DNA is bound by yNhp6A. Here, we employed an array of conformational probes (FRET, fluorescence anisotropy, and circular dichroism) to reveal solution conditions in which an 18- base-pair DNA oligomer indeed remains bound to yNhp6A while unbent. In 100 mM NaCl, yNhp6Abound DNA unbends as the temperature is raised, with no significant dissociation of the complex detected up to ∼45◦C. In 200 mM NaCl, DNA unbending in the intact yNhp6A complex is again detected up to ∼35◦C. Microseconds-resolved laser temperaturejump perturbation of the yNhp6a–DNA complex revealed relaxation kinetics that yielded unimolecular DNA bending/unbending rates on timescales of 500 s−1 ms. These data provide the first direct observation of bending/unbending dynamics of DNA in complex with yNhp6A, suggesting a bind-then-bend mechanism for this protein.
Department of Chemistry and Biochemistry
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UNI ScholarWorks, Rod Library, University of Northern Iowa
©2019 Manas Kumar Sarangi, Viktoriya Zvoda, Molly Nelson Holte, Nicole A. Becker, Justin Peters, L. James Maher III, and Anjum Ansari.
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Sarangi, Manas Kumar; Zvoda, Viktoriya; Holte, Molly Nelson; Becker, Nicole A.; Peters, Justin; Maher, L. James III; and Ansari, Anjum, "Evidence for a bind-then-bend mechanism for architectural DNA binding protein yNhp6A" (2019). Faculty Publications. 4.