Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast.

Timely completion of genome replication is a prerequisite for mitosis, genome integrity, and cell survival. A challenge to this timely completion comes from the need to replicate the hundreds of untranscribed copies of rDNA that organisms maintain in addition to the copies required for ribosome biogenesis. Replication of these rDNA arrays is relegated to late S phase despite their large size, repetitive nature, and essentiality. Here, we show that, in Saccharomyces cerevisiae, reducing the number of rDNA repeats leads to early rDNA replication, which results in delaying replication elsewhere in the genome. Moreover, cells with early-replicating rDNA arrays and delayed genome-wide replication aberrantly release the mitotic phosphatase Cdc14 from the nucleolus and enter anaphase prematurely. We propose that rDNA copy number determines the replication time of the rDNA locus and that the release of Cdc14 upon completion of rDNA replication is a signal for cell cycle progression.

The effects of manipulating levels of replication initiation factors on origin firing efficiency in yeast.

Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome.

Phenotypic and Genotypic Consequences of CRISPR/Cas9 Editing of the Replication Origins in the rDNA of Saccharomyces cerevisiae.

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, thus the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae, developed independently but similar to one developed by others. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9-mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication have on cellular health. We find that long-term growth of the edited clones results in faster-growing suppressors that have acquired segmental aneusomy of the rDNA-containing region of chromosome XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently, and homogeneously edit the rDNA origin via CRISPR/Cas9.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants.

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae.

A natural polymorphism in rDNA replication origins links origin activation with calorie restriction and lifespan.

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.

Natural polymorphism in BUL2 links cellular amino acid availability with chronological aging and telomere maintenance in yeast.

Aging and longevity are considered to be highly complex genetic traits. In order to gain insight into aging as a polygenic trait, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard strain RM11 and a laboratory strain S288c, to identify quantitative trait loci that control chronological lifespan. Among the major loci that regulate chronological lifespan in this cross, one genetic linkage was found to be congruent with a previously mapped locus that controls telomere length variation. We found that a single nucleotide polymorphism in BUL2, encoding a component of an ubiquitin ligase complex involved in trafficking of amino acid permeases, controls chronological lifespan and telomere length as well as amino acid uptake. Cellular amino acid availability changes conferred by the BUL2 polymorphism alter telomere length by modulating activity of a transcription factor Gln3. Among the GLN3 transcriptional targets relevant to this phenotype, we identified Wtm1, whose upregulation promotes nuclear retention of ribonucleotide reductase (RNR) components and inhibits the assembly of the RNR enzyme complex during S-phase. Inhibition of RNR is one of the mechanisms by which Gln3 modulates telomere length. Identification of a polymorphism in BUL2 in this outbred yeast population revealed a link among cellular amino acid availability, chronological lifespan, and telomere length control.