Mapping of the 3'-end positions of simian virus 40 nascent strands

Using the instability of replication loops as the basis for the isolation of replication origins, we have undertaken an analysis of the 3' ends of the extruded nascent strands of replicating simian virus 40 (SV40) DNA. DNA fragments containing the SV40 origin of replication were obtained by digesting highly purified replicative intermediates of SV40 with BamHI and then heating at 55 degrees C for 16h. The origin-containing fragments extruded under these conditions were purified and cloned into pBR322. We used restriction mapping to analyze 640 clones of the 674 that contained SV40 sequences. A large majority of the clones were found to contain rearrangements in the sequences of either pBR322 or SV40 and were disregarded. Those clones that contained legitimate SV40 and pBR322 sequences were presumed to have been derived from the extruded SV40 nascent strands and were further analyzed. A combination of restriction enzymes was used that allowed us to define the 3' ends with an accuracy of +/- 20 base-pairs. The results of restriction analysis were confirmed by nucleotide sequence analysis of selected clones. The results show that the replication forks move with a high degree of symmetry, with respect to the initiation site of DNA replication, and are consistent with the existence of pause sites for the extension of replication forks. From the clones analyzed, it appears that the center of the replication bubble is to the early side of the BglI site.