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.

Evolutionary adaptation increases the fitness of a species in its environment. It can occur through rewiring of gene regulatory networks, such that an organism responds appropriately to environmental changes. We investigated whether sirtuin deacetylases, which repress transcription and require NAD+ for activity, serve as transcriptional rewiring points that facilitate the evolution of potentially adaptive traits. If so, bringing genes under the control of sirtuins could enable organisms to mount appropriate responses to stresses that decrease NAD+ levels. To explore how the genomic targets of sirtuins shift over evolutionary time, we compared two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, that display differences in cellular metabolism and life cycle timing in response to nutrient availability. We identified sirtuin-regulated genes through a combination of chromatin immunoprecipitation and RNA expression. In both species, regulated genes were associated with NAD+ homeostasis, mating, and sporulation, but the specific genes differed. In addition, regulated genes in K. lactis were associated with other processes, including utilization of nonglucose carbon sources, detoxification of arsenic, and production of the siderophore pulcherrimin. Consistent with the species-restricted regulation of these genes, sirtuin deletion affected relevant phenotypes in K. lactis but not S. cerevisiae. Finally, sirtuin-regulated gene sets were depleted for broadly conserved genes, consistent with sirtuins regulating processes restricted to a few species. Taken together, these results are consistent with the notion that sirtuins serve as rewiring points that allow species to evolve distinct responses to low NAD+ stress.

To understand the process by which new protein functions emerge, we examined how the yeast heterochromatin protein Sir3 arose through gene duplication from the conserved DNA replication protein Orc1. Orc1 is a subunit of the origin recognition complex (ORC), which marks origins of DNA replication. In Saccharomyces cerevisiae, Orc1 also promotes heterochromatin assembly by recruiting the structural proteins Sir1-4 to silencer DNA. In contrast, the paralog of Orc1, Sir3, is a nucleosome-binding protein that spreads across heterochromatic loci in conjunction with other Sir proteins. We previously found that a non-duplicated Orc1 from the yeast Kluyveromyces lactis behaved like ScSir3 but did not have a silencer-binding function like ScOrc1. Moreover, K. lactis lacks Sir1, the protein that interacts directly with ScOrc1 at the silencer. Here, we examined whether the emergence of Sir1 coincided with Orc1 acting as a silencer-binding protein. In the non-duplicated species Torulaspora delbrueckii, which has an ortholog of Sir1 (TdKos3), we found that TdOrc1 spreads across heterochromatic loci independently of ORC, as ScSir3 and KlOrc1 do. This spreading is dependent on the nucleosome binding BAH domain of Orc1 and on Sir2 and Kos3. However, TdOrc1 does not have a silencer-binding function: T. delbrueckii silencers do not require ORC binding sites to function, and Orc1 and Kos3 do not appear to interact. Instead, Orc1 and Kos3 both spread across heterochromatic loci with other Sir proteins. Thus, Orc1 and Sir1/Kos3 originally had different roles in heterochromatin formation than they do now in S. cerevisiae.

5Evolutionary adaptation increases the fitness of an organism in its environment. It can occur through 6 rewiring of gene regulatory networks, such that an organism responds appropriately to environmental 7 changes. We investigated whether sirtuin deacetylases, which repress transcription and require NAD + 8 for activity, could facilitate the evolution of potentially adaptive responses by serving as transcriptional 9 rewiring points. If so, bringing genes under the control of sirtuins could enable organisms to mount 10 appropriate responses to stresses that decrease NAD + levels. To explore how the genomic targets of 11 sirtuins shift over evolutionary time, we compared two yeast species, Saccharomyces cerevisiae and 12 Kluyveromyces lactis that display differences in cellular metabolism and lifecycle timing in response to 13 nutrient availability. We identified sirtuin-regulated genes through a combination of chromatin 14 immunoprecipitation and RNA expression. In both species, regulated genes were associated with NAD + 15 homeostasis, mating, and sporulation, but the specific genes differed. In addition, regulated genes in K. 16 lactis were associated with other processes, including utilization of non-glucose carbon sources, heavy 17 metal efflux, DNA synthesis, and production of the siderophore pulcherrimin. Consistent with the 18 species-restricted regulation of these genes, sirtuin deletion impacted relevant phenotypes in K. lactis 19 but not S. cerevisiae. Finally, sirtuin-regulated gene sets were depleted for broadly-conserved genes, 20 consistent with sirtuins regulating processes restricted to a few species. Taken together, these results 21 are consistent with the notion that sirtuins serve as rewiring points that allow species to evolve distinct 22 responses to low NAD + stress. 23 4 59 BEDALOV et al. 2003). We compared the targets of ScSir2 and ScHst1 in S. cerevisiae with those of KlSir2 60 in K. lactis. This species did not undergo the duplication that led to Sir2 and Hst1. Like ScSir2, KlSir2 acts 61 at the telomeres, cryptic mating-type loci, and rDNA; and like ScHst1, KlSir2 acts at mid-sporulation 62 genes (ASTROM et al. 2000;HICKMAN AND RUSCHE 2009). 63 An unresolved question is whether the sets of genes repressed by Sir2 and Hst1 in S. cerevisiae and 64 K. lactis differ functionally, enabling each species to respond in a distinct way to low intracellular NAD + . 65 There are several key differences between these species that are connected to cellular metabolism and 66 nutrient availability. For example, in the presence of oxygen, S. cerevisiae processes sugars through 67 fermentation whereas K. lactis favors respiration (KIERS et al. 1998). These distinct metabolic strategies 68