Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo

Abstract

Initiation of simian virus 40 (SV40) DNA replication is facilitated by two auxiliary sequences that flank the minimally required origin (ori) core sequence. In monkey cells, the replication rate of each of the four ori configurations changed with time after transfection in a characteristic pattern. This pattern was reproduced in an extract from SV40-infected monkey cells by varying the ratio of DNA substrate to cell extract; DNA replication in vitro depended on ori auxiliary sequences to the same extent as they did in vivo. Facilitation by ori auxiliary sequences was lost at high ratios of DNA to cell extract, revealing that the activity of these sequences required either multiple initiation factors or a molar excess of one initiation factor bound to ori. This parameter, together with ionic strength and the method used to measure DNA replication, determined the level of facilitation by ori auxiliary sequences in vitro. The activity of ori auxiliary sequences was not diminished in vivo or in vitro by increasing amounts of large tumor antigen. Therefore, ori auxiliary sequences promoted initiation of replication at some step after tumor antigen binding to ori. Furthermore, although cellular factors could modulate the activity of ori auxiliary sequences in vitro, these factors did not appear to involve nucleosome assembly because no correlation was observed between the number of nucleosomes assembled per DNA molecule and facilitation by ori auxiliary sequences. These results demonstrate that SV40 ori auxiliary sequences can function in vitro as they do in vivo and begin to elucidate their role in initiating DNA replication.