We have used physical mapping techniques, based largely upon cleavage with restriction endonucleases, to examine the structure and mechanism of formation of unintegrated and integrated species of retroviral DNA in infected cells. Upon infection by ASV, a virus-coded DNA polymerase slowly catalyzes the synthesis of an unusual double-stranded (DS) linear DNA molecule in the cytoplasm. This DNA is approximately 300 bp longer than viral RNA, and it contains a redundant sequence of approximately 300 bases at its ends. The redundancy includes sequences specific to both the 3' and the 5' termini of viral RNA, and it is organized in a manner denoted 3'5'---3'5'. Construction of linear DNA requires that the DNA polymerase be twice transposed from one template to another. Furthermore, the minus strand (complementary to viral RNA) appears to be initiated by a single primer (tRNA(Trp)) and slowly elongated as a single strand; the second (plus) stands are made discontinuously but are initiated and terminated in a specific fashion. Upon transport to the nucleus, linear DNA is converted to at least two forms of circular DNA. Most of the monomeric circles contain only one copy of the 300-base redundant sequence and are presumably formed by intramolecular recombination between the termini of linear DNA. The rest of the monomeric circles contains two copies of the redundancy and is therefore about 300 bp larger than the smaller species; the mechanism by which the larger circles is formed is not known. In addition, small amounts of dimeric circles, of unknown function and origin, have been observed. Viral DNA can integrate into many sites in host cell DNA, but it is not known if the sites are selected at random. Proviral DNA in clones of ASV-transformed rat cells appears to be coextensive with unintegrated linear DNA (i.e., cell DNA-3'5'---3'5' cell DNA), creating a suitable template for synthesis of viral RNA, flanked by additional viral sequencies which could have regulatory functions. Although the immediate precursor to the integrated provirus is not yet known, integration of circular DNA would require recognition of a specific site in viral DNA by the enzymes that mediate integrative recombination. In addition, replication of cellular DNA seems to be necessary for integrative recombination.
|Number of pages||14|
|Journal||Cold Spring Harbor Symposia on Quantitative Biology|
|Publication status||Published - Dec 1 1979|
ASJC Scopus subject areas
- Molecular Biology