Open AccessInitiation of simian virus 40 DNA replication in vitro: pulse-chase experiments identify the first labeled species as topologically unwound.
Author(s) -
Peter A. Bullock,
YeonSoo Seo,
Jerard Hurwitz
Publication year - 1989
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.86.11.3944
Subject(s) - biology , dna , simian , dna replication , replication factor c , origin of replication , virus , microbiology and biotechnology , in vitro , virology , eukaryotic dna replication , genetics
A distinct unwound form of DNA containing the simian virus 40 (SV40) origin is produced in replication reactions carried out in mixtures containing crude fractions prepared from HeLa cells. This species, termed form UR, comigrates on chloroquine-containing agarose gels with the upper part of the previously described heterogeneous highly unwound circular DNA, form U. As with form U, formation of form UR is dependent upon the SV40 tumor (T) antigen. Pulse-chase experiments demonstrate that the first species to incorporate labeled deoxyribonucleotides comigrates with form UR. Restriction analyses of the products of the pulse-chase experiments show that initiation occurs at the SV40 origin and then proceeds outward in a bidirectional manner. These experiments establish form UR as the earliest detectable substrate for SV40 DNA replication and suggest that SV40 DNA replication initiates on an unwound species.