Bernd Otto

Deoxyribonucleic acid replication in vitro

Abstract We describe an in vitro system for DNA replication which uses a highly concentrated lysate of DNA polymerase I deficient Escherichia coli bacteria. The DNA synthesis observed in vitro proceeds over long periods of time, and the rates of total DNA synthesis and of chain, elongation are 10 to 20% of the in vivo rate. This in vitro DNA synthesis resembles in vivo replication in many aspects. It is semiconservative. Furthermore the DNA is synthesized in small pieces which become joined together upon prolonged incubation. Treatments which specifically inhibit in vivo replication affect the rate of in vitro synthesis. The rate is much reduced when the cells have been pretreated with ultraviolet light or mitomycin, when cells have been allowed to finish the replication cycle in the absence of further initiation, when synthesis takes place in the presence of nalidixic acid, or when synthesis takes place at non-permissive temperature in lysates of mutants that are temperature-sensitive with respect to DNA replication. DNA synthesis depends on the presence of soluble macromolecules other than DNA polymerase I or II. These molecules must be present at a concentration comparable to their in vivo concentration.

Maturation of newly replicated chromatin of simian virus 40 and its host cell

Abstract The DNA in replicating simian virus 40 chromatin and cellular chromatin was labeled with short pulses of [ 3 H]thymidine. The structure of pulse-labeled nucleoprotein complexes was studied by micrococcal nuclease digestion. It was found that in both newly replicated viral and cellular chromatin, a structural state appears which is characterized by an increased sensitivity to nuclease and a faster than usual rate of cleavage to DNA fragments of monomeric nucleosome size and smaller. Pulse-chase experiments show that each of these effects requires a characteristic time to disappear in both systems, suggesting the existence of different sub-processes of chromatin maturation. One of these processes, detectable by the reversion of the unusually fast production of subnucleosomal fragments, is delayed in SV40 chromatin replication.