Isolation of a Drosophila amplification origin developmentally activated by transcription

We exploited the Drosophila Amplicon in Follicle Cells, DAFC-62D, to identify a new metazoan amplification origin, ori62. In addition to the origin, DAFC-62D contains two other developmental stage-specific binding regions for the Origin Recognition Complex (ORC) and the replicative helicase MCM2-7. All three of these regions are required for proper amplification. There are two rounds of amplification initiation at ori62, and the second round is preceded by transcription across ori62. We show by alpha-amanitin inhibition that RNA polymerase II (RNAPII) transcription is required to localize MCM2-7 (but not ORC) to permit the second round of origin firing. This role for transcription appears unique to DAFC-62D, because neither other DAFCs nor ectopic transposons with the DAFC-62D replication elements bounded by functional chromatin insulators are affected by alpha-amanitin. By sequential chromatin immunoprecipitation, we show that the MCM complex and RNAPII are bound to the same 100-500 bp pieces of chromatin during late origin firing. These results raise the possibility that RNAPII may recruit MCM2-7 at some metazoan replication origins.