Biochemical investigations of control of replication initiation of plasmid R6K.
The mechanistic basis of control of replication initiation of plasmid R6K was investigated by addressing the following questions. What are the biochemical attributes of mutations in the pi initiator protein that caused loss of negative control of initiation? Did the primary control involve only initiator protein-ori DNA interaction or did it also involve protein-protein interactions between pi and several host-encoded proteins? Mutations at two different regions of the pi-encoding sequence individually caused some loss of negative control as indicated by a relatively modest increase in copy number. However, combinations of the mutation P42L, which caused loss of DNA looping, with those located in the region between the residues 106 and 113 induced a robust enhancement of copy number. These mutant forms promoted higher levels of replication in vitro in a reconstituted system consisting of 22 purified proteins. The mutant forms of pi were susceptible to pronounced iteron-induced monomerization in comparison with the WT protein. As contrasted with the changes in DNA-protein interaction, we found no detectable differences in protein-protein interaction between wild type pi with DnaA, DnaB helicase, and DnaG primase on one hand and between the high copy mutant forms and the same host proteins on the other. The DnaG-pi interaction reported here is novel. Taken together, the results suggest that both loss of negative control due to iteron-induced monomerization of the initiator and enhanced iteron-initiator interaction appear to be the principal causes of enhanced copy number.
Abhyankar
Mayuresh M
MM
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Reddy
Jagan M
JM
Sharma
Rahul
R
Büllesbach
Erika
E
Bastia
Deepak
D
D001426:Bacterial Proteins / Q000737:chemistry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry; D019915:DNA Primase / Q000737:chemistry; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry; D053488:DnaB Helicases; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism*; D011485:Protein Binding
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D009841:Oligonucleotides; D004247:DNA; D019915:DNA Primase; D004265:DNA Helicases; D053488:DnaB Helicases