Quantitative analysis of nucleotide modulation of DNA binding by DnaC protein of Escherichia coli.
In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form. DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E. coli DNA replication. In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin. We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC-DNA and DnaC-DnaB interactions using fluorescence anisotropy and affinity sensor analysis. DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides. DnaC bound ssDNA in the complete absence of nucleotides. The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP[S]. In the presence of ADP, the binding affinity decreased approximately fifty-fold. Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch. Both ssDNA and nucleotides modulate DnaB6*DnaC6 complex formation, which has significant implications in DnaC protein function. Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein.
Biswas
Subhasis B
SB
Department of Molecular Biology, School of Medicine, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Stratford, NJ 08084, USA. subhasis.biswas@umdnj.edu
Flowers
Stephen
S
Biswas-Fiss
Esther E
EE
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D016880:Anisotropy; D001426:Bacterial Proteins; D015374:Biosensing Techniques; D004265:DNA Helicases / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D008954:Models, Biological; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004265:DNA Helicases; D053488:DnaB Helicases