Synthesis of multimeric polyoma virus DNA in mouse L-cells: role of the tsA1S9 gene product

Abstract

Several different forms of progeny viral DNA can be identified in polyoma virus (Py)-infected mouse L-cells. The majority comprise mature form I superhelical DNA and the circular, double-stranded "theta" replicating intermediates in which the progeny DNA strands never exceed the unit genome length of the template. There is formed, in addition, a minority fraction of multimeric, linear, double-stranded Py DNA molecules that sediment heterogeneously at 28 to 35S and greater than 35S. Restriction enzyme analysis of these large Py DNA molecules reveals them to be tandem arrays of multiple unit genome lengths, covalently linked head to tail. It is estimated that the 28 to 35S multimeric DNA has an average size of about 20 megadaltons, made up of 6 to 20 Py genome units. The greater than 35S Py DNA is, of course, larger. Kinetic analysis indicates that formation of the monomeric progeny viral DNA and the 28 to 35S multimeric Py DNA reaches a peak at about 35 to 36 h postinfection. Synthesis of the very large linear molecules of greater than 35S is first detected after this interval and continues thereafter. The de novo synthesis of all of these progeny Py DNA molecules proceeds apparently normally in Py-infected tsA1S9 mouse L-cells incubated at 38.5 degrees C under conditions which restrict normal cellular DNA replication. These findings suggest that the cellular DNA topoisomerase II activity, encoded in the tsA1S9 locus (R. W. Colwill and R. Sheinin, submitted for publication), is not required for de novo formation of any form of Py DNA. However, the total amount made and the rate of synthesis of the large molecular weight Py DNA are affected very late in temperature-inactivated tsA1S9 cells.

DNA synthesis in Balb/C-3T3 ts 2 cells is restricted by a temperature-sensitive function of late G1 phase

“…The kinetics of heat inactivation of DNA synthesis in ts 2 cultures resemble, in a general way, those observed with dna,s/S,s mouse L-cells [cf. Sheinin (1976Sheinin ( , 1980, Sheinin & Guttman (1977), Setterfield et al (1978), Sheinin et al ( , 1978a, Guttman & Sheinin (1979), Sheinin & Lewis (1980), and Ganz & Sheinin (1983)]. Any DNA synthesis carried out by such cells incubated at the npt for an elapse time equivalent to two generation periods is effected by normal semiconservative, semidiscontinuous replication.…”