Only centromeres can supply the partition system required for ARS functionin the yeast Yarrowia lipolytica

Autonomously replicating sequences (ARSs) in the yeast Yarrowia lipolytica require two components: an origin of replication (ORI) and centromere (CEN) DNA, both of which are necessary for extrachromosomal maintenance. To inve stigate this cooperation in more detail, we performed a screen for genomic sequences able to confer high frequency of transformation to a plasmid-born e ORI. Our results confirm a cooperation between ORI and CEN sequences to f orm an ARS, since all sequences identified in this screen displayed feature s of centromeric DNA and included the previously characterized CEN1-1, CEN3 -1 and CEN5-1 fragments. Two new centromeric DNAs were identified as they a re unique, map to different chromosomes (II and TV) and induce chromosome b reakage after genomic integration. A third sequence, which is adjacent to, but distinct from the previously characterized CEN1-1 region was isolated f rom chromosome I. Although these CEN sequences do not share significant seq uence similarities, they display a complex pattern of short repeats, includ ing conserved blocks of 9 to 14 bp and regions of dyad symmetry. Consistent with their A + T-richness and strong negative roll angle, Y. lipolytica CE N-derived sequences, but not ORIs, were capable of binding isolated Drosoph ila nuclear scaffolds. However, a Drosophila scaffold attachment region tha t functions as an ARS in other yeasts was unable to confer autonomous repli cation to an ORI-containing plasmid. Deletion analysis of CEN1-1 showed tha t the sequences responsible for the induction of chromosome breakage could be eliminated without compromising extrachromosomal maintenance. We propose that, while Y, lipolytica CEN DNA is essential for plasmid maintenance, th is function can be supplied by several sub-fragments which, together, form the active chromosomal centromere. This complex organization of Y. lipolyti ca centromeres is reminiscent of the regional structures described in the y east Schizosaccharomyces pombe or in multicellular eukaryotes. :(C), 2001 A cademic Press.