Mutational analysis of DNA sequences affecting the replication of defective polyomavirus variant D-50.

The genome of the defective polyomavirus variant D-50 consists of tandemly repeated DNA segments. The repeat unit corresponds to a 17% fragment of polyomavirus DNA (Griffin and Fried (1975) Nature 256, pp. 175-179). To allow mutational analysis, a monomer unit of D-50 DNA was cloned. After excision from the plasmid and ligation to form a random mixture of products, circular head to tail oligomers of the cloned segment were replicated in transfected cells. Those molecules had the same replication properties as original D-50 DNA. Deletion of base sequences assumed to be at the origin of DNA synthesis inhibited the replication completely, whereas a deletion of a segment at the junction of the tandem repeats had only a slight inhibitory effect. Mutation of potential coding sequences of the variant genome had a slight stimulatory effect on DNA synthesis, ruling out that D-50 expresses any protein that stimulates replication. In an attempt to construct variants similar to D-50, cells were transfected with polyomavirus DNA fragments including the sequences of the D-50 monomer unit. However, all these molecules replicated very slowly, suggesting the presence of an element that inhibited DNA synthesis. The combined data show that D-50 genomes can be reconstituted by ligation of monomer units and that the origin of DNA replication was the only essential element of the variant genome, whereas other elements had cis-acting auxiliary functions.