Identification of the site of interruption in relaxed circles producing during bacteriophage lambda DNA circle replication
R C Reuben, A Skalka
Journal of Virology
The DNA that accumulates in a X infection restricted to the early (circular) stage of replication consists of approximately two-thirds covalently closed circles and one-third relaxed circles bearing a single interruption in either strand of the duplex. The latter molecules are presumed to be a unique class in that the interruption is not repairable by DNA polymerase and ligase. Preferential radioisotopic labeling of the region immediately adjacent to the interruption, followed by hybridization
... o sheared fragments of the X chromosome with varying guanine plus cytosine content, suggested that the nick resides at the position of the mature molecular ends of the X chromosome. Digestion of the labeled molecules with restriction enzymes and reconstruction experiments in which Hershey circles were generated by annealing of interrupted strands isolated from the relaxed circles support this interpretation. The results indicate that the relaxed circles consist of a population containing one interruption in either of the two strands of the duplex jointly representing the two "nicks" contained in Hershey circles (in which the cohesive ends are annealed). These molecules could result from the inability of the maturation function to make the required staggered endonucleolytic cuts when the DNA substrate is a monomeric circle rather than a multimeric linear molecule. Alternatively, this interruption could be the result of an endonucleolytic cutting event critical to DNA replication. Bacteriophage X replicates its chromosome in prevents the transition to rolling-circle replicatwo distinct stages. Upon infection of the host tion and is capable of degrading any linear cell the linear duplex chromosome circularizes concatemers that result from rolling-circle repby means of its single-stranded cohesive ends lication (13). Mutations in the phage and host and undergoes a limited number of rounds of general recombination systems (red and recA, circle replication (L. Enquist and A. Skalka, respectively) prevent residual maturation by unpublished observations). There then ensues a blocking the formation of multimeric DNA moltransition to a rolling-circle mode of replication ecules via recombinational pathways. The cirwhereby multimeric linear "concatemers" are cular DNA molecules that accumulate in the produced. The concatemer structure is required absence of the gamma, red, and recA proteins for maturation of phage DNA and packaging are not efficiently packaged even though head into mature phage particles (4, 6, 7, 29-31).