Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication

Site-specific fluorescence double-labeling of proteins and analysis of structural changes in solution by Fluorescence Resonance Energy Transfer (FRET)

“…The DnaA BA recombinant plasmids were fully sequenced in both directions by Eurofins MWG Operon LLC (Louisville, KY) and verified to be free of any fortuitous mutation(s) before expression and purification. The detailed procedure for expression and purification of proteins is discussed separately [ 7 ].…”

“…The multiplication factor was chosen based upon trial and error to show the best normalized spectrum. After selection of this methodology, we used this algorithm for all of the data transformations through the remainder of our study [ 7 ]. Bulk FRET data can be corrected with either Fluortools or Microsoft Excel as discussed above.…”

“…4 , the graphs were compiled in a master spreadsheet to plot the normalized spectra. Individual spectrum of the double-labeled protein with or without substrate(s) were then selected and presented to show the structural changes in the protein with changes in the FlAsH and Alexa 568 emission peaks [ 7 ]. The spectra produced by GraphPad Prism were smoothened using the feature provided by the program.…”

Identification of the Unwinding Region in the Clostridioides difficile Chromosomal Origin of Replication

“…DnaA boxes are typically 9-mer non-palindromic DNA sequences, and the Escherichia coli DnaA box consensus sequence is TTWTNCACA ( Schaper and Messer, 1995 ; Wolanski et al, 2014 ). The boxes can differ in their affinity for DnaA, and even demonstrate different dependencies on the ATP co-factor ( Speck et al, 1999 ; Patel et al, 2017 ). Binding of Domain IV to the DnaA boxes promotes higher-order oligomerization of DnaA, forming a filament that wraps around DNA ( Erzberger et al, 2006 ; Ozaki et al, 2012 ; Scholefield and Murray, 2013 ).…”

“…Binding of Domain IV to the DnaA boxes promotes higher-order oligomerization of DnaA, forming a filament that wraps around DNA ( Erzberger et al, 2006 ; Ozaki et al, 2012 ; Scholefield and Murray, 2013 ). It is thought that the interaction of the DnaA filament with the DNA helix introduces a bend in the DNA ( Erzberger et al, 2006 ; Patel et al, 2017 ). The resulting superhelical torsion facilitates the melting of the adjacent [A + T]-rich DNA Unwinding Element (DUE) ( Kowalski and Eddy, 1989 ; Erzberger et al, 2006 ; Zorman et al, 2012 ).…”