GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast <i>Pichia pastoris</i>

<div><p>The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, <i>Pichia pastoris</i>, simultaneously employs at least two types of replication origins—a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect <i>P. pastoris</i> origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in <i>P. pastoris</i> and imply an evolutionary bifurcation of the modes of replication initiation.</p></div

Replication timing of the <i>P. pastoris</i> genome.

<p>(A) Genomic DNA from G1 and S phase cells was sheared and sequenced. Normalized S/G1 DNA copy ratios (in 1 kbp windows) were smoothed and plotted against chromosomal coordinates. Peaks correspond to positions of replication initiation. The profile of chromosome 4 is shown (all chromosomes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004169#pgen.1004169.s006" target="_blank">Figure S6</a>) with ARS locations indicated by open (AT-ARSs) and shaded (GC-ARSs) circles. Un-smoothed ratio data for one of the replicates is shown are grey. Coordinates of replication timing peaks are indicated by dashed vertical lines. (B) The distributions of smoothed S/G1 ratio data. The distribution of all ratios (“Genome”) is shown adjacent to the distribution of values at bins containing midpoints of GC-ACSs (“GC”) or AT-ARSs (“AT”). Values for ARSs that have no other ARSs within 40 kb in both directions are shown on the right (“isolated”). (C) The complete genomic ratio distribution is shown relative to distributions after removal of data within 60 kb ranges centered on AT-ARSs (“AT”), GC-ARSs (“GC”), or all ARSs (“all ARS”). (D) For each ARS, the distance to the nearest replication peak was calculated. The ARS-peak distances are shown as distributions separately for GC-ARSs (blue) and AT-ARSs (orange). Peak distances from simulated random sets of loci are shown in grey.</p

The GC-ACS is required for GC-ARS function.

<p>Wild type (WT) and mutant (MUT) alleles of the twelve ARSs indicated were cloned into a <i>URA3</i> ARS-less vector and used to transform <i>ura3</i> yeast on selective medium plates lacking uracil. Plates were grown at 30°C for five days before pictures were taken. Colony formation indicates plasmid maintenance and ARS activity. The GC-ACS was positioned <15 bp away from the 5′ endpoint in all ARS sequences. The sequences of the fragments tested are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004169#pgen.1004169.s012" target="_blank">Table S4</a>.</p