Abstract
An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described. Mu replication was monitored by DNA hybridization. Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific. Mu DNA synthesis is semidiscontinuous. In the presence of the DNA ligase inhibitor NMN, about one-half of the DNA was in Okazaki pieces and one-half in large DNA. The Mu Okazaki pieces hybridized mainly to the Mu light strand; the large DNA hybridized mainly to the Mu heavy strand. Okazaki pieces isolated from uninfected cells also hybridized to 2000-3000 bases of host DNA present in Mu-separated strands. However, the host Okazaki pieces hybridize to both Mu strands symmetrically. Most, if not all, host sequences were represented in mature Mu viral DNA. The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right.
Highlights
From the §Department of Biology, University of Utah, Salt Lake City, Utah 84112 and the tDepartment of Biochemistry, University of Wyoming, Laramie, Wyoming 82071
DNA replication on cellophane dISCS allows host replication forks to continue in vitro with characteristics that approximate those in vivo
Using lysates of HB101::Mucts, DNA synthesis on the disc was linear for a longer period compared to a nonlysogen (Fig. 1); a 30-45% increase in incorporation was usually observed at 30 min
Summary
From the §Department of Biology, University of Utah, Salt Lake City, Utah 84112 and the tDepartment of Biochemistry, University of Wyoming, Laramie, Wyoming 82071. An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described. Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific. Okazaki pieces isolated from uninfected cells hybridized to 2000-3000 bases of host DNA present in Mu-separated strands. The host Okazaki pieces hybridize to both Mu strands symmetrically. The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right. The life cycle of the bacteriophage Mu [1] is unusual in that during vegetative growth, replication is coupled to transposition. If a Mu lysogen is induced, transposition of the viral genome to over a hundred sites in the chromosome takes place in less than 1 h. The high transposition frequency makes Mu a specially attractive system for biochemical study
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