Abstract
By using an in vitro system for R1 plasmid replication dependent on a plasmid-encoded repA protein and host dnaA protein, 5' ends of the nascent leading strand were located at positions 1986-1992, some 380 base pair downstream of oriR. Analyses of early replication intermediates generated in vitro in the presence of dideoxy TTP also indicated that replication initiates about 400 base pair downstream of oriR and proceeds unidirectionally. When a 418-base single-stranded DNA from position 1778 to 2195, derived from the leading strand template, was cloned onto an M13 vector, the chimeric single-stranded phage could be replicated in vitro with only single-stranded DNA binding protein, primase (dnaG gene product), and DNA polymerase III holoenzyme. Furthermore, the priming occurred at a site identical to leading strand initiation. These results strongly suggest that the leading strand synthesis is primed by primase alone. The lagging strand synthesis is specifically terminated at position 1515 or 1516 within oriR, preventing further leftward fork movement. Based on these results, a scheme of R1 plasmid replication is presented.
Highlights
By using an in vitrosystem for R1 plasmid replication dependent on a plasmid-encodedrepA protein and hostdnaA protein, 5’ ends of the nascent leading strand were located at positions 1986-1992, some 380 base pair downstreamof oriR
With purified proteins was conducted in 25 pl of assay mixtures containing Hepes.KOH, 40mM, potassium glutamate, 40 mM; magnesium acetate, 10 mM; glycerol, 16%;dithiothreitol, 4 mM; bovine serum albumin, 80 pg/ml; ATP, 1.6mM; CTP,GTP, and UTP, 0.4 mM each; dATP, dCTP, dGTP,and TTP, 80 p~ each with. These results show that replication intermediates are accumulated in vitro in thepresence of dideoxythymidine triphosphates (ddTTP)
Half of the digested materialswere nascent DNA fragments with 5’ ends around position 1986 further treated with alkaline as described under “Materials are generated by de novo synthesis and not by processing of longer fragments
Summary
-72 fragment, chimeric single-stranded phage DNAs containing each strand derived downstream from oriR were immobilized onto a nitrocellulose filter and were hybridized with the 32P-. Alkaline treatment of the products resulted in decrease of the sizes of the fragments by one to two nucleotides (Fig. 3B), suggestingthe presence of RNA primer attached to its 5‘ end Half of the digested materialswere nascent DNA fragments with 5’ ends around position 1986 further treated with alkaline (lane 2 ) as described under “Materials are generated by de novo synthesis and not by processing of longer fragments. These fragments were synthesized in a very early stage of and Methods.”.
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