Purification and properties of Escherichia coli protein i, a prepriming protein in phi X174 DNA replication.
Protein i, one of seven Escherichia coli proteins essential for primosome initiation of DNA chains in the in vitro conversion of single-stranded phi X174 DNA to duplex replicative form, has been purified approximately 15,000-fold to more than 98% purity. The protein is an oligomer of 22,000-dalton subunits migrating as a single electrophoretic band on native, as well as on denaturing polyacrylamide gels. Estimates of a Stokes radius of 41 A, a sedimentation coefficient of 3.5 S, a Mr = 61,000, and a frictional coefficient of 1.57 suggest that native protein i is a highly asymmetric oligomer composed of three identical subunits. About 50 such molecules are present/cell. Cross-linking the protein with dimethylsuberimidate or dimethyladipimidate produced three major bands corresponding to the monomer, dimer, and trimer, as well as two minor bands corresponding to the tetramer and pentamer. Incorporation of 3H-labeled "trimeric" protein i into the prepriming replication intermediate (primosome) occurs at a stage requiring participation of dnaB and dnaC proteins, and follows the actions of proteins n, n', and n". After extension of primers by DNA polymerase III holoenzyme, protein i is not retained in the isolated primosome complex. Thus, protein i is essential in the assembly of a functional primosome, but its precise physiologic role and genetic locus are still unknown.
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010584:Bacteriophage phi X 174 / Q000201:enzymology*; D003432:Cross-Linking Reagents; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008970:Molecular Weight; D014779:Virus Replication
D000596:Amino Acids; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D029968:Escherichia coli Proteins; C017685:protein I, E coli