10.1371/journal.pgen.1006561.g008
Katrin Schenk
Katrin
Schenk
Ana B. Hervás
Ana B.
Hervás
Thomas C. Rösch
Thomas C.
Rösch
Marc Eisemann
Marc
Eisemann
Bernhard A. Schmitt
Bernhard A.
Schmitt
Stephan Dahlke
Stephan
Dahlke
Luise Kleine-Borgmann
Luise
Kleine-Borgmann
Seán M. Murray
Seán
M. Murray
Peter L. Graumann
Peter
L. Graumann
DnaAE183Q mutant shows reduced ATPase activity and an underinitiating phenotype.
Public Library of Science
2017
half time binding
initiation control
replication origin regions
residence time
affinity binding sites
chromosomal binding sites
ORC
DNA replication DnaA
FRAP
2017-02-06 19:05:05
Figure
https://plos.figshare.com/articles/figure/DnaAE183Q_mutant_shows_reduced_ATPase_activity_and_an_underinitiating_phenotype_/4624459
<p>A) Coupled ATPase assay of purified wild type DnaA or of DnaAE183Q (10 μM). B) Surface plasmon resonance [SPR] experiments with DnaA and DnaAE183Q [2.5 μM] binding to <i>oriC</i>-DNA [0.25 pmol]. Black line: DnaAE183Q, grey line: wtDnaA. C) SPR in the presence of 2.5 mM ATP. D-E) Fluorescence microscopy analysis of cells growing exponentially in rich (LB) medium, D) expressing YFP-DnaAE183Q for 60 minutes, E) expressing YFP-DnaA for 60 minutes. Note that cells contain more <i>oriC</i> regions in rich medium compared with minimal medium as in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006561#pgen.1006561.g001" target="_blank">1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006561#pgen.1006561.g002" target="_blank">2</a>. The effect of induction of mutant DnaA is more apparent in rich medium. Septa are indicated by white lines, and are more difficult to see after induction of mutant DnaA. F)-I) Increased cell size and reduced number of origins in exponentially growing cells expressing DnaAE183Q for 60 min in minimal medium, white bars wild type cells, grey bars cells after induction of DnaAE183Q, F) Diagram showing the distribution of cell length, bars show percentage of cells with a certain size, white bars wild type cells, G) Diagram showing percentage of cells containing one or two nucleoids, or none, H) Diagram showing the percentage of cells containing a certain number of origins, I) Diagram showing the percentage of cells containing no YFP-DnaA foci, or different numbers of foci.</p>