Analysis of regions essential for the function of chromosomal replicator sequences from Yarrowia lipolytica

Two DNA segments exhibiting ARS (autonomously replicating sequence) activity in the dimorphic yeast Yarrowia lipolytica were cloned from its chromosome on an integrative LEU2 plasmid. These ARS segments, designated Y1ARS1 and Y1ARS2, conferred on the hybrid plasmids high transformation efficiency and enabled extrachromosomal transmission of the plasmids in 1 or 2 copies per yeast cell under selective conditions. Deletion analysis showed that at least 728-1003 bp for Y1ARS1 and 1377-1629 bp for Y1ARS2 were required for full function. Both of these regions contained two 10/11 matches to an ARS core consensus in Saccharomyces cerevisiae, whereas neither was similar to the S. cerevisiae centromere sequence. Significantly, both Y1ARS elements contained at, or close to, their boundaries a 13 bp sequence, 5'-TATATTCAAGCAA-3', which resembles the cleavage site for topoisomerase II in Drosophila. A central 524 bp ClaI fragment of Y1ARS2 contained four stretches of a 17 bp direct repeat sequence, 5'-GAAAAACAAAAACAGGC-3', and exhibited the electrophoretic behavior typical of bent DNA.