Gene duplications increase organismal robustness by providing freedom for gene divergence or by increasing gene dosage. The yeast histone chaperones Fpr3 and Fpr4 are paralogs that can assemble nucleosomes in vitro; however, the genomic locations they target and their functional relationship is poorly understood. We refined the yeast synthetic genetic array approach to enable the functional dissection of gene paralogs. Applying this method to Fpr3 and Fpr4 uncovered redundant, cooperative, and divergent functions. While Fpr3 is uniquely involved in chromosome segregation, Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. We find that the TRAMP5 RNA exosome is critical for fitness in Δfpr3Δfpr4 yeast and leverage this information to identify an important role for Fpr4 at the 5′ ends of protein coding genes. Additionally, Fpr4 and TRAMP5 negatively regulate RNAs from the nontranscribed spacers of ribosomal DNA. Yeast lacking Fpr3 and Fpr4 exhibit a genome instability phenotype at the ribosomal DNA, which implies that these histone chaperones regulate chromatin structure and DNA access at this location. Taken together. we provide genetic and transcriptomic evidence that Fpr3 and Fpr4 operate separately, cooperatively, and redundantly to regulate a variety of chromatin environments.

The nucleoplasmin family of histone chaperones is identified by a pentamer-forming domain and multiple acidic tracts that mediate histone binding and chaperone activity. Within this family, a novel domain organization was recently discovered that consists of an N-terminal nucleoplasmin-like (NPL) domain and a C-terminal FKBP peptidyl-proline isomerase domain. Saccharomyces cerevisiae Fpr4 is one such protein. Here we report that in addition to its known histone prolyl isomerase activities, the Fpr4 FKBP domain binds to nucleosomes and nucleosome arrays in vitro. This ability is mediated by a collection of basic patches that enable the enzyme to stably associate with linker DNA. The interaction of the Fpr4 FKBP with recombinant chromatin complexes condenses nucleosome arrays independently of its catalytic activity. Based on phylogenetic comparisons we propose that the chromatin binding ability of ‘basic’ FKBPs is shared amongst related orthologues present in fungi, plants, and insects. Thus, a subclass of FKBP prolyl isomerase enzymes is recruited to linker regions of chromatin.

In both prokaryotes and eukaryotes, hydroxyurea is suggested to inhibit DNA replication by inactivating ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. In this study, we show that the inhibition of replication in is transient even at concentrations of 0.1 M hydroxyurea and that replication rapidly recovers and continues in its presence. The recovery of replication does not require the alternative ribonucleotide reductases, NrdEF and NrdDG, or translesion DNA polymerases, Pol II, Pol IV, or Pol V. Ribonucleotides are incorporated at higher frequencies during replication in the presence of hydroxyurea. However, these do not contribute significantly to the observed synthesis or toxicity. Hydroxyurea toxicity was only observed under conditions where the stability of hydroxyurea was compromised and byproducts, known to damage DNA directly, were allowed to accumulate. The results demonstrate that hydroxyurea is not a direct or specific inhibitor of DNA synthesis, and that the transient inhibition observed is most likely due to a general depletion of iron cofactors from enzymes when 0.1 M hydroxyurea is initially applied. Finally, the results support previous studies suggesting that hydroxyurea toxicity is mediated primarily through direct DNA damage induced by the breakdown products of hydroxyurea, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell. Hydroxyurea is commonly suggested to function by inhibiting DNA replication through the inactivation of ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. Here, we show that hydroxyurea only transiently inhibits replication in before it rapidly recovers and continues in the presence of this drug. The recovery of replication does not depend on alternative ribonucleotide reductases, translesion synthesis, or RecA. Further we show that hydroxyurea toxicity is only observed after toxic intermediates that accumulate when hydroxyurea breaks down, damage DNA and induce lethality. The results demonstrate that hydroxyurea toxicity is mediated indirectly by the formation of DNA damage, rather than by an inhibition of replication or depletion of deoxyribonucleotide levels in the cell.

Gene duplications increase organismal robustness by providing freedom for gene divergence or by increasing gene dosage. The yeast histone chaperones Fpr3 and Fpr4 are paralogs that can assemble nucleosomesin vitro, however the genomic locations they target and their functional relationship is poorly understood. We refined the yeast synthetic genetic array (SGA) approach to enable the functional dissection of gene paralogs. Applying this method to Fpr3 and Fpr4 uncovered their redundant and divergent functions: while Fpr3 is uniquely involved in chromosome segregation, Fpr3 and Fpr4 co-operate on some genes and are redundant on others where they impact gene expression and transcriptional processivity. We find that the TRAMP5 RNA exosome is essential inΔfpr3Δfpr4yeast and leverage this information to identify Fpr3/4 target loci. Amongst these are the non-transcribed spacers of ribosomal DNA where either paralog is sufficient to establish chromatin that is both transcriptionally silent and refractory to recombination. These data provide evidence that Fpr3 and Fpr4 have shared chromatin-centric functions, especially at nucleolar rDNA. However, their distinct genetic interaction profiles show they also have evolved separate functions outside of the nucleolus.