INITIATION OF SV40 DNA-REPLICATION IN-VITRO - ANALYSIS OF THE ROLE PLAYED BY SEQUENCES FLANKING THE CORE ORIGIN ON INITIAL SYNTHESIS EVENTS

Replication initiation events are suppressed over the SV40 core origin in vitro; they are also greatly reduced over sequences flanking the o rigin which contain binding sites for several transcription factors. T o address the biochemical basis for the gap in initiation events over the flanking sequences, initial synthesis events have been characteriz ed on templates lacking these sequences. Herein, it is demonstrated th at previously functional initiation sites are nearly inactive when mov ed to positions that are proximal to the core origin. Thus, the gap in initiation events depends, in part, on the proximity of the initiatio n sites to the SV40 core origin. Additional experiments demonstrate th at removal of the flanking sequences had little or no effect on DNA un winding or on the efficiency of initiation of DNA synthesis in vitro. These results indicate that, under our in vitro conditions, initiation of SV40 DNA synthesis is not enhanced by binding of transcription fac tors to the flanking sequences. (C) 1997 Academic Press