Abstract
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.
Highlights
Protein i, one of seven Escherichia coli proteins essential for primosome initiation of DNA chains in the in vitro conversion of single-stranded 4X174 DNA to duplex replicative form, has been purified -15,000-fold to more than 98% purity
Understanding how each of these proteins contributes to the formation of this complex will help to clarify the mechanism of initiation of Okazaki fragments in the discontinuous phase of replication of the E. coli chromosome
We describe the purification to near homogeneity of protein i, one of these E. coli proteins involved in the prepriming reaction on OX DNA, and some functional and physicochemical properties of the protein
Summary
One of seven Escherichia coli proteins essential for primosome initiation of DNA chains in the in vitro conversion of single-stranded 4X174 DNA to duplex replicative form, has been purified -15,000-fold to more than 98% purity. After extension of primers by DNA polymerase m holoenzyme, protein i is not retained in the isolated primosome complex. Conversion of the single-stranded chromosomes of the three small bacteriophages, M13, G4, and pX174, to their duplex replicative forms differ primarily in the mode of initiation of priming replication § Present address, Department of Chemistry, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan. ¶ Present address, Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205. We describe the purification to near homogeneity of protein i, one of these E. coli proteins involved in the prepriming reaction on OX DNA, and some functional and physicochemical properties of the protein
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