The proposed research seeks to determine the involvement of the DNA relaxing enzyme in simian virus 40 (SV40) and eukaryotic DNA replication. SV40 DNA replication can serve as a model for cellular DNA replication since, like cellular DNA, SV40 DNA is replicated in the nucleus by cellular enzymes and is associated with cellular histones to form the characteristic nucleosome structure. SV40 chromosomes isolated at low ionic strength from infected monkey cells retain the ability to replicate DNA in vitro when supplied with deoxy- and ribonucleoside triphoshates. Thus, at least some of the enzymes which participate in in vivo DNA replication must be bound to the replicating chromosome (RC). The DNA relaxing enzyme cosediments with RC (and also with non-RC) on neutral glycerol gradients and thus may be bound to RC and participate in their replication. Attempts to determine if the enzyme is bound by further purifying the RC have thus far been unsuccessful. As an alternative approach, the requirement of the enzyme for in vitro SV40 DNA synthesis will be tested after addition of either a specific inhibitor (if one can be found) or anti-relaxing enzyme antibodies (if they can be raised towards the purified enzyme). A 125,000 molecular weight form of the enzyme has been purified from nuclei of bovine liver in the presence of protease inhibitor. The previously characterized enzyme of approximately 64,000 molecular weight appears to be an active proteolytic fragment of the larger species. Attempts are being made to eliminate the variable proteolytic degradation and to isolate and characterize the intact enzyme.