Origins of replication act as sites for the initiation of DNA replication via recruitment of ORC (Origin Recognition Complex). ORC mediates loading of the MCM2-7 helicase to ‘license’ replication competence. Although ORC is conserved in eukaryotes, the mechanism for origin recognition is not. S. cerevisiae ORC binds to a conserved sequence motif present at all origins. S. pombe and metazoan ORCs binds non-specifically to AT-rich DNA. Genome-wide studies have uncovered several general features of origins.
We are developing a high throughput DNA sequencing approach to obtain a comprehensive map of replication initiation sites in Tetrahymena thermophila. This approach will provide the first comprehensive picture of origin usage in the early diverging Ciliophora lineage and reveal genomic features of replication origins. As part of this project, we have developed a functional assay to validate replication origins. Our data indicate that specific intergenic DNA sequences are required to support DNA replication of episomal plasmids as autonomously replicating sequences (ARSs). ARS assays are being used to map origins and uncover DNA sequences requirements. To identify new origins, nine non-coding segments from a small non-rDNA macronuclear chromosome were introduced into an origin-less vector that confers resistance to paromomycin. These plasmids were introduced into vegetative Tetrahymena by biolistic transformation as a pool. A plasmid containing the well-characterized rDNA origin served as positive control, and an empty vector served as negative control. The non-rDNA pool generated >100 folds more transformants compared to negative controls. After cultured for >100 generations, episomal DNA was recovered by Qiagen columns and re-transformed into E. coli. Massive DNA rearrangements were detected in E. coli clones. This was not unexpected, as AT-rich Tetrahymena sequence frequently rearrange in E.coli. Thus far we have recovered sequences from one 0.8 kb Tetrahymena insert, suggesting that this fragment supports DNA replication in Tetrahymena. We are currently use direct sequencing to identify Tetrahymena origin sequences. As further confirmation, endogenous chromosomal loci are being examined by 2D gel electrophoresis.
Our lab recently discovered an unconventional DNA replication program in Tetrahymena that operates when ORC proteins are dramatically depleted. Known origins are silenced and cryptic origins are activated. This suggests the presence of cryptic origins whose chromosomal replication initiation signal is too weak to be detected in population-based assays. We plan to exploit ARS assays to identify replication determinants under high and low ORC conditions.