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by 2D gel analysis to have weak origin activity, whereas other proORis that functioned
as ARSs lacked origin function. In the case of P. pastoris, analysis of ARS function on plasmids identified two separate ARS sequences, one class was a GC-rich sequence that when mutated eliminated ARS activity and a separate class with AT-rich sequences233.
A representative of both classes was shown to colocalize with origin activity in the chromosomal context. In both the studies with C. albicans and P. pastoris, the biochemical role of the conserved sequences was not addressed. DNA sequences associated with ARS activity have been shown to either bind the initiator protein ORC, exclude nucleosomes
or bind to other proteins that mediate ARS plasmid stability, such as proteins that tether plasmids to the nuclear periphery234. It is also possible that the GC-rich ARSs in P. pastoris overlap with gene promoters and these may contribute to origin activity. Thus, future studies of ARS activity in yeasts must address the biochemical function of any proposed conserved sequences to reveal how they contribute to ARS or origin activity, which may not be equivalent.
5.1 Co-evolutionary transitions of origin specificity, gene silencing mechanisms and centromeres
In species that lack DNA sequence-specific origins of replication, inherited transcriptional gene silencing can occur by RNA interference (RNAi)141. These species also have epigenetically defined centromeres (CEN)235. A group of intermediate branching yeasts seem to be in the transition of losing RNAi (e.g., C. albicans236; Figure 4), since they still carry some but not all genes that encode RNAi proteins (or non-canonical Dicer gene). The most recently branching budding yeasts, including S. cerevisiae (Figure 4), have completely lost RNAi and instead, they have gained ORC-Sir4 mediated gene silencing. This has been accompanied by the acquisition of the Orc4 α-helix, the Orc2 DNA interacting loop and sequence specific origins141 and a DNA sequence-specific, point CEN configuration235.
5.2 Evolutionary driving forces
Previously, it has been suggested that acquiring and maintaining a beneficial killer virus could explain the loss of RNAi in budding yeast236. In addition, the loss of RNAi
and the acquisition of sequence-specific point centromeres were suggested to co-evolve with maintenance of the circular 2-micron plasmid, which is known as a selfish DNA element that uses the host segregation apparatus components for plasmid stability235. It was proposed that DNA sequence-defined, point centromeres were acquired by integration of the selfish circular 2-micron plasmid into the genome, bringing with it DNA sequences that eventually functioned as centromeres235. RNAi contributes to the silencing and stability of repetitive DNA sequences like heterochromatin satellite repeated sequences at centromeres and remnants of transposable elements, as well as organization of rDNA repeats237.
The S. cerevisiae and K. lactis (Figure 4) clade of budding yeasts have lost centromere- associated repeated sequences and have replaced RNAi with Silent Information Regulator (SIR) proteins that silence gene transcription in a DNA sequence-specific manner at the silent mating type loci, as well as maintaining rDNA and telomere repeats by preventing recombination238. Interestingly, the acquisition of the Sir4 protein that binds either directly to ORC in K. lactis or indirectly via Sir1 to ORC in S. cerevisiae, occurred with the acquisition of the Orc4 α-helix and the Orc2 loop, both of which contribute to the DNA