Origin remodeling and opening in bacteria rely on distinct assembly states of the DnaA initiator.
The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage single-stranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single- and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.
Duderstadt
Karl E
KE
Biophysics Graduate Group, California Institute for Quantitative Biology, University of California, Berkeley, California 94720, USA.
Mott
Melissa L
ML
Crisona
Nancy J
NJ
Chuang
Kevin
K
Yang
Haw
H
Berger
James M
JM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D021122:Protein Subunits