GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris
(A) Schematic of ARS-seq and miniARS-seq screens. Fragmented genomic DNA was ligated into non-replicating URA3 vectors and screened for ARS activity followed by deep sequencing of the resultant plasmid inserts (ARS-seq, top). ARS-seq plasmid inserts were amplified and sheared using DNase I. Short fragments of ARSs were ligated into the URA3 vectors and screened for ARS activity followed by deep sequencing of the plasmid inserts (miniARS-seq, bottom). (B) The GC-ACS motif identified by the MEME algorithm. (C) The distribution of MAST motif scores of the best match to the GC-ACS in every PpARS. (D) 2D gel analysis at loci A2772 (putative AT-ARS at chromosome 1: 2,772 kb) and C379 (putative GC-ARS at chromosome 3: 379 kb). The red arrows highlight arcs corresponding to replication bubble intermediates.