The effects of transcription and RNA processing on the initiation of chloroplast DNA replication in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, the origin for chloroplast DNA replication, Ori A. overlaps the coding region for the chloroplast ribosomal protein Rp116. In an in vitro DNA replication system that uses cloned Ori A as template, alteration of transcription across rpl16 affects replication activity. S1 nuclease protection mapping of cellular RNA derived from this region revealed multiple 5' and 3' ends. and several 3' ends were mapped within mini Ori A (224 bp), the core region for replication initiation. We also demonstrated that the protein fraction used in the in vitro DNA replication system contained an RNA processing activity responsible for the generation of multiple 3' ends. The 3' ends of some of the processed RNA species coincided with those of the cellular transcripts. Initiation of DNA replication in the in vitro system changed the abundance of some of the processed RNA species, and the S1 nuclease protection pattern generated by the 3' ends now mimicked that of the in vivo transcripts. We also monitored the pattern of 3' ends in cellular transcripts from the rpl16 region during gametogenesis - when the chloroplast DNA is under-replicated - and detected a change in transcript abundance that correlated with that seen in the in vitro study. Measurements of the template activity of mutants with targeted sequences change near the sites of processing also supported the notion that the processed transcripts play an important role in DNA replication