Analysis of the biased distribution of topoisomerase I break sites on replicating simian virus 40 DNA.
We previously mapped the locations of breaks introduced by eukaryotic topoisomerase I (topo I) in replicating simian virus 40 (SV40) DNA and observed an approximate 3:1 bias in the distribution of the break sites for the template strand for discontinuous DNA synthesis. In the present study, this bias has been confirmed by the mapping of additional sites utilizing a standard primer extension assay and a sensitive repetitive primer extension (RPE) method. No new sites could be detected on either strand of SV40 by the RPE method, despite the 10 to 20 fold greater sensitivity of the technique. To investigate the nature of the bias, a detailed analysis of the SV40 DNA sequence was undertaken. A set of 17 pentanucleotide sequences derived from those sites observed to be broken in the viral DNA extracted from SV40-infected cells define an in vivo consensus sequence. We show that the observed strand bias is likely due to the intrinsic asymmetric distribution of these consensus sequences on the two strands of SV40 DNA. To confirm these observations, double-stranded oligonucleotides containing previously identified in vivo topo I break sites were introduced in both orientations into SV40 to generate insertion mutants. Mapping experiments utilizing these mutants revealed that the inserted topo I break sites were broken in vivo regardless of their orientation, confirming that the SV40 sequence is the major, if not the sole determinant, of the observed strand bias. The possible origins of the strand bias are discussed in relation to the evolution of the virus.
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
L H
LH
Parker
J J
JJ
Champoux
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D012091:Repetitive Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D004264:DNA Topoisomerases, Type I