THE ORIGIN RECOGNITION COMPLEX HAS ESSENTIAL FUNCTIONS IN TRANSCRIPTIONAL SILENCING AND CHROMOSOMAL REPLICATION

The role of the origin recognition complex (ORC) was investigated in replication initiation and in silencing. Temperature-sensitive mutations in ORC genes caused defects in replication initiation at chromosomal origins of replication, as measured by two-dimensional (2-D) origin-mapping gels, fork migration analysis, and plasmid replication studies. These data were consistent with ORC functioning as a eukaryotic replication initiator. Some origins displayed greater replication initiation deficiencies in ore mutants than did others, revealing functional differences between origins. Alleles of ORC5 were isolated that were defective for silencing but not replication, indicating that ORC's role in silencing could be separated from its role in replication. In temperature-sensitive ore mutants arrested in mitosis, temperature-shift experiments caused a loss of silencing, indicating both that ORC had functions outside of the S phase of the cell cycle and that ORC was required for the maintenance of the silenced state.