Conservation of a DNA Replication Motif among Phylogenetically Distant Budding Yeast Species

Abstract

Eukaryotic DNA replication begins at genomic loci termed origins, which are bound by the Origin Recognition Complex (ORC). Although ORC is conserved across species, the sequence composition of origins is more varied. In the budding yeast Saccharomyces cerevisiae, the ORC binding motif consists of an A/T-rich 17 bp “extended ACS” sequence adjacent to a B1 element composed of two 3 bp motifs. Similar sequences occur at origins in closely related species, but it is not clear when this type of replication origin arose and whether it pre-dated a whole genome duplication that occurred around 100 million years ago in the budding yeast lineage. To address these questions, we identified the ORC binding sequences in the non-duplicated species Torulaspora delbrueckii. We used chromatin immunoprecipitation followed by sequencing and identified 190 ORC binding sites distributed across the eight T. delbrueckii chromosomes. Using these sites, we identified an ORC binding motif that is nearly identical to the known motif in S. cerevisiae. We also found that the T. delbrueckii ORC binding sites function as origins in T. delbrueckii when cloned onto a plasmid and that the motif is required for plasmid replication. Finally, we compared an S. cerevisiae origin with two T. delbrueckii ORC-binding sites and found that they conferred similar stabilities to a plasmid. These results reveal that the ORC-binding motif arose prior to the whole genome duplication and has been maintained for over 100 million years.

The DNA replication protein Orc1 from the yeast Torulaspora delbrueckii is required for heterochromatin formation but not as a silencer-binding protein

“…Signal from input chromatin is shown in black. (C) Association of Orc1-Myc at HMR2 in sir2Δ (red) and kos3Δ (green) backgrounds and distribution of Orc2-V5, and Orc4-V5 at HMR2 (M aria et al . 2021).…”

“…We also sequenced input chromatin as a control for regions over-represented in the starting material. For comparison, we examined previously published ChIP-Seq data for Sir2, Sir4, Orc2 and Orc4 (E llahi and R ine 2016; M aria et al . 2021).…”

“…All growth media were supplemented with geneticin to select for plasmids. (F) Distribution at HMR2 of wild-type Orc1-Myc (LRY3211, blue), orc1-bahΔ- Myc (LRY3212, light blue), orc1-P179A -Myc (LRY3215, teal), Orc2-V5, and Orc4-V5 (M aria et al . 2021).…”

“…The recognition sequences for Rap1, Abf1, and ORC were predicted at each of the mating-type loci (Table 1) and are indicated by colored boxes in schematics of chromosomal features. For ORC, genome-wide binding sites and motifs were previously reported (M aria et al . 2021).…”

“…Endogenous 3-phosphoglycerate kinase (Pgk1) was detected as a loading control. (C-F) The distribution of TdOrc1-Myc (LRY3211), TdKos3-V5 (LRY3211), TdSir2-V5 and TdSir4-V5 (E llahi and R ine 2016), and TdOrc2-V5 and TdOrc4-V5 (M aria et al . 2021) at (C) a replication origin ARS-VII69, (D) HMR1 , (E) HMR2 , and (F) HML were determined using chromatin immunoprecipitation followed by sequencing (ChIP-Seq).…”