Topoisomerase I sites cluster asymmetrically at the ends of the simian virus 40 core origin of replication.
In vivo, topoisomerase I cleavage sites are located predominantly on the strands of simian virus 40 DNA that are the templates for discontinuous synthesis (S.E. Porter and J.J. Champoux, Mol. Cell. Biol. 9:541-550, 1989). This arrangement of sites suggests that topoisomerase I may associate with replication complexes in unique functional orientations at replication forks. We have mapped topoisomerase I cleavage sites in the simian virus 40 origin of replication in vitro under conditions suitable for DNA replication. Numerous sites cluster in the inverted repeat and AT-rich domains at the ends of the core origin and are arranged on the same strands that are cut most frequently in vivo. We propose that cleavage at these sites would allow bidirectional extension of the replication bubble induced by T antigen within the core origin of replication early in the initiation of DNA synthesis. A mutational analysis of the topoisomerase I sites confirms the importance of positions -4 to -1 and +1 in the consensus sequence 5'-A/T-A/G-A/T-T-break-G/A-3'. Surprisingly, more distant nucleotide positions also influence topoisomerase I sites in the inverted repeat and AT-rich domains of the core origin. The effects of distant sequences could be mediated by direct interactions with topoisomerase I or by the conformation of DNA in the core origin.
Tsui
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Department of Microbiology, State University of New York, Stony Brook 11794-8621.
Anderson
M E
ME
Tegtmeyer
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P
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics*; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology
D004279:DNA, Viral; D004264:DNA Topoisomerases, Type I