Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites.

Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA) array. Each rDNA repeat contains a programmed replication fork barrier (RFB) established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1∆ and fob1∆ similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth.

Rnr1, but not Rnr3, facilitates the sustained telomerase-dependent elongation of telomeres.

Ribonucleotide reductase (RNR) provides the precursors for the generation of dNTPs, which are required for DNA synthesis and repair. Here, we investigated the function of the major RNR subunits Rnr1 and Rnr3 in telomere elongation in budding yeast. We show that Rnr1 is essential for the sustained elongation of short telomeres by telomerase. In the absence of Rnr1, cells harbor very short, but functional, telomeres, which cannot become elongated by increased telomerase activity or by tethering of telomerase to telomeres. Furthermore, we demonstrate that Rnr1 function is critical to prevent an early onset of replicative senescence and premature survivor formation in telomerase-negative cells but dispensable for telomere elongation by Homology-Directed-Repair. Our results suggest that telomerase has a "basal activity" mode that is sufficient to compensate for the "end-replication-problem" and does not require the presence of Rnr1 and a different "sustained activity" mode necessary for the elongation of short telomeres, which requires an upregulation of dNTP levels and dGTP ratios specifically through Rnr1 function. By analyzing telomere length and dNTP levels in different mutants showing changes in RNR complex composition and activity we provide evidence that the Mec1ATR checkpoint protein promotes telomere elongation by increasing both dNTP levels and dGTP ratios through Rnr1 upregulation in a mechanism that cannot be replaced by its homolog Rnr3.

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment.

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance.

Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that several SAMHD1 mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of one SAMHD1 allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associated SAMHD1 mutations increase mutation rates in cancer cells.

The mutation spectrum in genomic late replication domains shapes mammalian GC content.

Genome sequence compositions and epigenetic organizations are correlated extensively across multiple length scales. Replication dynamics, in particular, is highly correlated with GC content. We combine genome-wide time of replication (ToR) data, topological domains maps and detailed functional epigenetic annotations to study the correlations between replication timing and GC content at multiple scales. We find that the decrease in genomic GC content at large scale late replicating regions can be explained by mutation bias favoring A/T nucleotide, without selection or biased gene conversion. Quantification of the free dNTP pool during the cell cycle is consistent with a mechanism involving replication-coupled mutation spectrum that favors AT nucleotides at late S-phase. We suggest that mammalian GC content composition is shaped by independent forces, globally modulating mutation bias and locally selecting on functional element. Deconvoluting these forces and analyzing them on their native scales is important for proper characterization of complex genomic correlations.

Determination of deoxyribonucleoside triphosphate concentrations in yeast cells by strong anion-exchange high-performance liquid chromatography coupled with ultraviolet detection.

DNA polymerase assays are commonly used for the detection of deoxyribonucleoside triphosphates (dNTPs) in biological samples. For better specificity and accuracy, high-performance liquid chromatography (HPLC) methods have been developed for the analysis of the four dNTPs in complex samples. Here we describe a simple method using isocratic strong anion-exchange (SAX) chromatographic separation coupled with ultraviolet detection (UV) for the analysis of the four dNTPs in budding yeast Saccharomyces cerevisiae. This method can be applied to other species of yeast or bacteria.

Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer.

The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific.

Ribonucleotides incorporated during DNA replication are removed by RNase H2-dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired ribonucleotides, initiating their removal, but this is accompanied by RNA-DNA-damage phenotypes. Here we show that these phenotypes are incurred by a high level of ribonucleotides incorporated by a leading strand-replicase variant, DNA polymerase (Pol) ɛ, but not by orthologous variants of the lagging-strand replicases, Pols α or δ. Moreover, loss of both RNases H1 and H2 is lethal in combination with increased ribonucleotide incorporation by Pol ɛ but not by Pols α or δ. Several explanations for this asymmetry are considered, including the idea that Top1 incision at ribonucleotides relieves torsional stress in the nascent leading strand but not in the nascent lagging strand, in which preexisting nicks prevent the accumulation of superhelical tension.

H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly.

The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.

Telomere length kinetics assay (TELKA) sorts the telomere length maintenance (tlm) mutants into functional groups.

Genome-wide systematic screens in yeast have uncovered a large gene network (the telomere length maintenance network or TLM), encompassing more than 400 genes, which acts coordinatively to maintain telomere length. Identifying the genes was an important first stage; the next challenge is to decipher their mechanism of action and to organize then into functional groups or pathways. Here we present a new telomere-length measuring program, TelQuant, and a novel assay, telomere length kinetics assay, and use them to organize tlm mutants into functional classes. Our results show that a mutant defective for the relatively unknown MET7 gene has the same telomeric kinetics as mutants defective for the ribonucleotide reductase subunit Rnr1, in charge of the limiting step in dNTP synthesis, or for the Ku heterodimer, a well-established telomere complex. We confirm the epistatic relationship between the mutants and show that physical interactions exist between Rnr1 and Met7. We also show that Met7 and the Ku heterodimer affect dNTP formation, and play a role in non-homologous end joining. Thus, our telomere kinetics assay uncovers new functional groups, as well as complex genetic interactions between tlm mutants.

Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA.

RNase H2-dependent ribonucleotide excision repair (RER) removes ribonucleotides incorporated during DNA replication. When RER is defective, ribonucleotides in the nascent leading strand of the yeast genome are associated with replication stress and genome instability. Here, we provide evidence that topoisomerase 1 (Top1) initiates an independent form of repair to remove ribonucleotides from genomic DNA. This Top1-dependent process activates the S phase checkpoint. Deleting TOP1 reverses this checkpoint activation and also relieves replication stress and genome instability in RER-defective cells. The results reveal an additional removal pathway for a very common lesion in DNA, and they imply that the "dirty" DNA ends created when Top1 incises ribonucleotides in DNA are responsible for the adverse consequences of ribonucleotides in RNase H2-defective cells.

Telomere length homeostasis responds to changes in intracellular dNTP pools.

Telomeres, the ends of linear eukaryotic chromosomes, shorten due to incomplete DNA replication and nucleolytic degradation. Cells counteract this shortening by employing a specialized reverse transcriptase called telomerase, which uses deoxyribonucleoside triphosphates (dNTPs) to extend telomeres. Intracellular dNTP levels are tightly regulated, and perturbation of these levels is known to affect DNA synthesis. We examined whether altering the levels of the dNTP pools or changing the relative ratios of the four dNTPs in Saccharomyces cerevisiae would affect the length of the telomeres. Lowering dNTP levels leads to a modest shortening of telomeres, while increasing dNTP pools has no significant effect on telomere length. Strikingly, altering the ratio of the four dNTPs dramatically affects telomere length homeostasis, both positively and negatively. Specifically, we find that intracellular deoxyguanosine triphosphate (dGTP) levels positively correlate with both telomere length and telomerase nucleotide addition processivity in vivo. Our findings are consistent with in vitro data showing dGTP-dependent stimulation of telomerase activity in multiple organisms and suggest that telomerase activity is modulated in vivo by dGTP levels.

Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype.

The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis.

Migratory passerine birds as reservoirs of Lyme borreliosis in Europe.

To define the role of birds as reservoirs and disseminators of Borrelia spirochetes, we characterized tick infestation and reservoir competence of migratory passerine birds in Sweden. A total of 1,120 immature Ixodes ricinus ticks were removed from 13,260 birds and assayed by quantitative polymerase chain reaction (PCR) for Borrelia, followed by DNA sequencing for species and genotype identification. Distributions of ticks on birds were aggregated, presumably because of varying encounters with ticks along migratory routes. Lyme borreliosis spirochetes were detected in 160 (1.4%) ticks. Borrelia garinii was the most common species in PCR-positive samples and included genotypes associated with human infections. Infestation prevalence with infected ticks was 5 times greater among ground-foraging birds than other bird species, but the 2 groups were equally competent in transmitting Borrelia. Migratory passerine birds host epidemiologically important vector ticks and Borrelia species and vary in effectiveness as reservoirs on the basis of their feeding behavior.