Electron Microscopic Analysis of Patterns of DNA Replication in the Presence of Cycloheximide

DNA from synchronously replicating nuclei of Physarum polycephalum was studied electron microscopically after 15, 30, 60, and 90 or 120 min of replication in the presence or absence of the protein synthesis inhibi tor cycloheximide. The repl icat ion-loop size-distribution showed that replication fork progression is severely retarded in the presence of cycloheximide. Analysis of repl ication-loop frequency showed a similar pattern in control and cyclo-heximidetreated samples, with an increase from 15 to 30 and 60 min. This suggests, surprisingly, that init iations of new replicons either may not be inhibi ted by cycloheximide or, alternatively, that all init iations have already taken place at the very start of S-phase. The latter conclusion is favored in the light of previous results in our laboratory, discussed here. We have previously suggested that the genome of Physarum polycephalum replicates as sets of replicons of size around 2-4 X 107 daltons (5-10 gm), occurring in temporal clusters of 3-4 (1). This model, arrived at by analysis of alkaline sucrose sedimentation patterns, was verified by electron microscope and autoradiographic analysis of replicating DNA (2). An earlier study on the effects of inhibition of protein synthesis with cycloheximide during S-phase suggested the following model for control of DNA-replication in Physarum (3): at least 10 temporal sets of replication units depend for their consecutive initiations on ongoing protein synthesis, while replicons once initiated are no longer protein-synthesis dependent. Evans et al. (5) concluded, however, that cycloheximide did not interfere with initiation of new replication units, while elongation of initiated progeny strands was inhibited. In our previous study on the effects of inhibition of protein synthesis on DNA replication (4), we found that the chain length increase of new DNA strands was slowed or prevented at three levels: (a) formation of Okazaki fragments, (b) jo in ing of Okazaki fragments to replicon size DNA, and (c) maturation (joining) of replicon size DNA to high molecular weight DNA. It is possible that the latter two effects could be a consequence of the first. This result, as well as that of Evans et al. (5), appears to be at variance with the results of Muldoon et al. (3). In this communication we have analyzed DNA-replication in the presence and absence of the protein synthesis inhibitor, cycloheximide, using electron microscopy. The main purpose of this study was to shed light on the question of whether fork progression (replication rate) is unaffected or slowed or stopped in the presence of cycloheximide. We also hoped to be able to reveal whether new initiations can occur in the absence of