Gap filling and not replication fork progression is the rate-limiting step in the replication of UV-damaged simian virus 40 DNA.

摘要

We have analyzed the structural characteristics of simian virus 40 replicative intermediate DNA produced after UV irradiation and the kinetics of conversion of this intermediate DNA into form I DNA. Replicative intermediate DNA isolated at 30 or 60 min after UV irradiation consists primarily of two species of molecules that sediment in neutral sucrose gradients as either Cairns theta structures or relaxed monomeric circles. Replication forks on the Cairns intermediate DNA are symmetrically located with respect to the origin of replication, ruling out the possibility of asymmetric pauses or blocks to replication fork progression at damage sites. The relaxed circles contain at least one randomly located discontinuity in the daughter strand. Pulse-chase experiments demonstrated that a UV fluence-dependent fraction of the Cairns intermediate DNA progresses through the relaxed circular intermediate before being converted to completed form I molecules. Disappearance of Cairns intermediate DNA occurs at the same rate in irradiated and unirradiated cells, whereas completion of the relaxed circular intermediate DNA occurs at a slow rate, relatively independent of UV fluence. These data support a model for replication of UV-damaged DNA in which replication rapidly continues past damage sites via a gap formation event.