DNA replication initiates at specific sites in chromosomes termed Origins of Replication (ORI). In eukaryotes, these sites are chosen by the binding of the Origin Recognition Complex (ORC) and subsequent recruitment of the MCM2-7 helicase to generate the pre-replicative complex during the G1 phase of the cell cycle. The relative contributions of the DNA sequence (cis-acting determinants) and chromatin context (histone modifications) to ORI function vary widely in eukaryotes, and are the subject of considerable debate. Previous studies revealed a role for the monomethylation of histone H3 on the 27th lysine residue (H3K27) in the control of DNA replication in the polyploid macronucleus of the model eukaryote, Tetrahymena thermophila (Gao et al., Genes Dev. 2013). H3K27 monomethylation appears to be restricted to the ciliate lineage and plants, and is mediated in Tetrahymena by TXR1 - a homologue of Arabidopsis thaliana ATXR5/ATXR6. TXR1 contains a SET-domain enabling the monomethylation of H3K27, and harbors a PCNA-interacting-protein (PIP) box. Taken together, the features of TXR1 suggest a role in DNA replication and/or DNA damage repair.
Using 2D gel electrophoresis of DNA replication intermediates (RIs), we provide evidence for the role of TXR1 in replication initiation in the ribosomal DNA (rDNA) minichromosome. Aberrantly migrating RIs emanate from the rDNA 5’ non-transcribed spacer (5’ NTS), but not from the rRNA coding region which is consistent with TXR1-dependent modification of intergenic histone H3 subunits. The novel properties of these RIs are unprecedented suggesting an unconventional nucleic-acid structure. To unveil the structure and nucleic acid content of aberrant RIs, we are employing different nuclease digestions and using the RNA-DNA hybrid-specific S9.6 monoclonal antibody to assess whether non-coding transcripts are involved. Finally, DNA fiber imaging of non-rDNA chromosomes revealed a global effect on replication initiation illustrated by the firing of fewer origins resulting in increased inter-origin distances in TXR1 knockout strains. The average rate for replication fork elongation in TXR1 mutants is indistinguishable from wild type; however, our data suggests that a subpopulation of forks either stall, collapse, or move slower in a TXR1 knockout background. We surmise two possibilities to explain the data: (1) TXR1-dependent modification of the Tetrahymena epigenome determines replication initiation sites, and (2) TXR1 is required to re-start stalled replication forks near origin initiation sites.