Publications by authors named "Andrei Chabes"

66 Publications

Proofreading deficiency in mitochondrial DNA polymerase does not affect total dNTP pools in mouse embryos.

Nat Metab 2020 08 10;2(8):673-675. Epub 2020 Aug 10.

Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

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http://dx.doi.org/10.1038/s42255-020-0264-zDOI Listing
August 2020

Elimination of rNMPs from mitochondrial DNA has no effect on its stability.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14306-14313. Epub 2020 Jun 8.

Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden;

Ribonucleotides (rNMPs) incorporated in the nuclear genome are a well-established threat to genome stability and can result in DNA strand breaks when not removed in a timely manner. However, the presence of a certain level of rNMPs is tolerated in mitochondrial DNA (mtDNA) although aberrant mtDNA rNMP content has been identified in disease models. We investigated the effect of incorporated rNMPs on mtDNA stability over the mouse life span and found that the mtDNA rNMP content increased during early life. The rNMP content of mtDNA varied greatly across different tissues and was defined by the rNTP/dNTP ratio of the tissue. Accordingly, mtDNA rNMPs were nearly absent in mice that have increased dNTP pools. The near absence of rNMPs did not, however, appreciably affect mtDNA copy number or the levels of mtDNA molecules with deletions or strand breaks in aged animals near the end of their life span. The physiological rNMP load therefore does not contribute to the progressive loss of mtDNA quality that occurs as mice age.
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http://dx.doi.org/10.1073/pnas.1916851117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322039PMC
June 2020

SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP.

Cell Rep 2020 05;31(6):107640

Medical Research Council Human Immunology Unit, Medical Research Council Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK. Electronic address:

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.
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http://dx.doi.org/10.1016/j.celrep.2020.107640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225753PMC
May 2020

High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase.

Nucleic Acids Res 2020 05;48(8):4274-4297

The Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.
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http://dx.doi.org/10.1093/nar/gkaa103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192613PMC
May 2020

Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication.

Mol Cell 2020 05 12;78(3):396-410.e4. Epub 2020 Mar 12.

Institut de Génétique Humaine, CNRS, Université de Montpellier, Montpellier, France. Electronic address:

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.
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http://dx.doi.org/10.1016/j.molcel.2020.02.021DOI Listing
May 2020

Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio.

Nucleic Acids Res 2020 01;48(1):264-277

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg D-69120, Germany.

The accumulation of mutations is frequently associated with alterations in gene function leading to the onset of diseases, including cancer. Aiming to find novel genes that contribute to the stability of the genome, we screened the Saccharomyces cerevisiae deletion collection for increased mutator phenotypes. Among the identified genes, we discovered MET7, which encodes folylpolyglutamate synthetase (FPGS), an enzyme that facilitates several folate-dependent reactions including the synthesis of purines, thymidylate (dTMP) and DNA methylation. Here, we found that Met7-deficient strains show elevated mutation rates, but also increased levels of endogenous DNA damage resulting in gross chromosomal rearrangements (GCRs). Quantification of deoxyribonucleotide (dNTP) pools in cell extracts from met7Δ mutant revealed reductions in dTTP and dGTP that cause a constitutively active DNA damage checkpoint. In addition, we found that the absence of Met7 leads to dUTP accumulation, at levels that allowed its detection in yeast extracts for the first time. Consequently, a high dUTP/dTTP ratio promotes uracil incorporation into DNA, followed by futile repair cycles that compromise both mitochondrial and nuclear DNA integrity. In summary, this work highlights the importance of folate polyglutamylation in the maintenance of nucleotide homeostasis and genome stability.
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http://dx.doi.org/10.1093/nar/gkz1006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145683PMC
January 2020

Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria.

Mol Cell 2019 12 3;76(5):784-796.e6. Epub 2019 Oct 3.

Department of Medical Biochemistry and Cell Biology, University of Gothenburg, PO Box 440, Gothenburg 405 30, Sweden. Electronic address:

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.
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http://dx.doi.org/10.1016/j.molcel.2019.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900737PMC
December 2019

dNTP production is essential for normal postnatal murine heart development.

J Biol Chem 2019 11 12;294(44):15889-15897. Epub 2019 Jul 12.

Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden

The building blocks of DNA, dNTPs, can be produced or can be salvaged from deoxyribonucleosides. However, to what extent the absence of dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the production of dNTPs, at embryonic day 13. All other tissues had normal dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in WT postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in WT hearts.
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http://dx.doi.org/10.1074/jbc.RA119.009492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827297PMC
November 2019

Ribonucleotides in mitochondrial DNA.

FEBS Lett 2019 07 24;593(13):1554-1565. Epub 2019 May 24.

Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.

The incorporation of ribonucleotides (rNMPs) into DNA during genome replication has gained substantial attention in recent years and has been shown to be a significant source of genomic instability. Studies in yeast and mammals have shown that the two genomes, the nuclear DNA (nDNA) and the mitochondrial DNA (mtDNA), differ with regard to their rNMP content. This is largely due to differences in rNMP repair - whereas rNMPs are efficiently removed from the nuclear genome, mitochondria lack robust mechanisms for removal of single rNMPs incorporated during DNA replication. In this minireview, we describe the processes that determine the frequency of rNMPs in the mitochondrial genome and summarise recent findings regarding the effect of incorporated rNMPs on mtDNA stability and function.
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http://dx.doi.org/10.1002/1873-3468.13440DOI Listing
July 2019

A geographically matched control population efficiently limits the number of candidate disease-causing variants in an unbiased whole-genome analysis.

PLoS One 2019 27;14(3):e0213350. Epub 2019 Mar 27.

Department of Medical Biochemistry and Biophysics, Umeå University, SE Umeå, Sweden.

Whole-genome sequencing is a promising approach for human autosomal dominant disease studies. However, the vast number of genetic variants observed by this method constitutes a challenge when trying to identify the causal variants. This is often handled by restricting disease studies to the most damaging variants, e.g. those found in coding regions, and overlooking the remaining genetic variation. Such a biased approach explains in part why the genetic causes of many families with dominantly inherited diseases, in spite of being included in whole-genome sequencing studies, are left unsolved today. Here we explore the use of a geographically matched control population to minimize the number of candidate disease-causing variants without excluding variants based on assumptions on genomic position or functional predictions. To exemplify the benefit of the geographically matched control population we apply a typical disease variant filtering strategy in a family with an autosomal dominant form of colorectal cancer. With the use of the geographically matched control population we end up with 26 candidate variants genome wide. This is in contrast to the tens of thousands of candidates left when only making use of available public variant datasets. The effect of the local control population is dual, it (1) reduces the total number of candidate variants shared between affected individuals, and more importantly (2) increases the rate by which the number of candidate variants are reduced as additional affected family members are included in the filtering strategy. We demonstrate that the application of a geographically matched control population effectively limits the number of candidate disease-causing variants and may provide the means by which variants suitable for functional studies are identified genome wide.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213350PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436687PMC
December 2019

The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity.

Nucleic Acids Res 2019 05;47(8):3986-3995

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA.

The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication.
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http://dx.doi.org/10.1093/nar/gkz048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486559PMC
May 2019

A recurrent cancer-associated substitution in DNA polymerase ε produces a hyperactive enzyme.

Nat Commun 2019 01 22;10(1):374. Epub 2019 Jan 22.

Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA.

Alterations in the exonuclease domain of DNA polymerase ε (Polε) cause ultramutated tumors. Severe mutator effects of the most common variant, Polε-P286R, modeled in yeast suggested that its pathogenicity involves yet unknown mechanisms beyond simple proofreading deficiency. We show that, despite producing a catastrophic amount of replication errors in vivo, the yeast Polε-P286R analog retains partial exonuclease activity and is more accurate than exonuclease-dead Polε. The major consequence of the arginine substitution is a dramatically increased DNA polymerase activity. This is manifested as a superior ability to copy synthetic and natural templates, extend mismatched primer termini, and bypass secondary DNA structures. We discuss a model wherein the cancer-associated substitution limits access of the 3'-terminus to the exonuclease site and promotes binding at the polymerase site, thus stimulating polymerization. We propose that the ultramutator effect results from increased polymerase activity amplifying the contribution of Polε errors to the genomic mutation rate.
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http://dx.doi.org/10.1038/s41467-018-08145-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343027PMC
January 2019

A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance.

Nucleic Acids Res 2019 01;47(1):237-252

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg D-69120, Germany.

The balance and the overall concentration of intracellular deoxyribonucleoside triphosphates (dNTPs) are important determinants of faithful DNA replication. Despite the established fact that changes in dNTP pools negatively influence DNA replication fidelity, it is not clear why certain dNTP pool alterations are more mutagenic than others. As intracellular dNTP pools are mainly controlled by ribonucleotide reductase (RNR), and given the limited number of eukaryotic RNR mutations characterized so far, we screened for RNR1 mutations causing mutator phenotypes in Saccharomyces cerevisiae. We identified 24 rnr1 mutant alleles resulting in diverse mutator phenotypes linked in most cases to imbalanced dNTPs. Among the identified rnr1 alleles the strongest mutators presented a dNTP imbalance in which three out of the four dNTPs were elevated (dCTP, dTTP and dGTP), particularly if dGTP levels were highly increased. These rnr1 alleles caused growth defects/lethality in DNA replication fidelity-compromised backgrounds, and caused strong mutator phenotypes even in the presence of functional DNA polymerases and mismatch repair. In summary, this study pinpoints key residues that contribute to allosteric regulation of RNR's overall activity or substrate specificity. We propose a model that distinguishes between different dNTP pool alterations and provides a mechanistic explanation why certain dNTP imbalances are particularly detrimental.
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http://dx.doi.org/10.1093/nar/gky1154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326808PMC
January 2019

Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage.

EMBO J 2018 11 29;37(21). Epub 2018 Aug 29.

Institut de Génétique Humaine, CNRS, Equipe Labellisée Ligue contre le Cancer, Université de Montpellier, Montpellier, France

The S-phase checkpoint maintains the integrity of the genome in response to DNA replication stress. In budding yeast, this pathway is initiated by Mec1 and is amplified through the activation of Rad53 by two checkpoint mediators: Mrc1 promotes Rad53 activation at stalled forks, and Rad9 is a general mediator of the DNA damage response. Here, we have investigated the interplay between Mrc1 and Rad9 in response to DNA damage and found that they control DNA replication through two distinct but complementary mechanisms. Mrc1 rapidly activates Rad53 at stalled forks and represses late-firing origins but is unable to maintain this repression over time. Rad9 takes over Mrc1 to maintain a continuous checkpoint signaling. Importantly, the Rad9-mediated activation of Rad53 slows down fork progression, supporting the view that the S-phase checkpoint controls both the initiation and the elongation of DNA replication in response to DNA damage. Together, these data indicate that Mrc1 and Rad9 play distinct functions that are important to ensure an optimal completion of S phase under replication stress conditions.
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http://dx.doi.org/10.15252/embj.201899319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213276PMC
November 2018

A mechanism for preventing asymmetric histone segregation onto replicating DNA strands.

Science 2018 09 16;361(6409):1386-1389. Epub 2018 Aug 16.

Institute for Cancer Genetics, Department of Pediatrics and Genetics and Development, Columbia University, New York, NY 10032, USA.

How parental histone (H3-H4) tetramers, the primary carriers of epigenetic modifications, are transferred onto leading and lagging strands of DNA replication forks for epigenetic inheritance remains elusive. Here we show that parental (H3-H4) tetramers are assembled into nucleosomes onto both leading and lagging strands, with a slight preference for lagging strands. The lagging-strand preference increases markedly in budding yeast cells lacking Dpb3 and Dpb4, two subunits of the leading strand DNA polymerase, Pol ε, owing to the impairment of parental (H3-H4) transfer to leading strands. Dpb3-Dpb4 binds H3-H4 in vitro and participates in the inheritance of heterochromatin. These results indicate that different proteins facilitate the transfer of parental (H3-H4) onto leading versus lagging strands and that Dbp3-Dpb4 plays an important role in this poorly understood process.
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http://dx.doi.org/10.1126/science.aat8849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597248PMC
September 2018

Rtt105 functions as a chaperone for replication protein A to preserve genome stability.

EMBO J 2018 09 31;37(17). Epub 2018 Jul 31.

Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly , but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.
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http://dx.doi.org/10.15252/embj.201899154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120668PMC
September 2018

Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling.

Genes Dev 2018 06 13;32(11-12):822-835. Epub 2018 Jun 13.

Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York 14853, USA.

The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of "free" Mec1 activation domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator proteins, "free" MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1's role in genome maintenance is separable from a previously unappreciated proreplicative function. Both Mec1's functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.
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http://dx.doi.org/10.1101/gad.308148.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049512PMC
June 2018

Upregulation of dNTP Levels After Telomerase Inactivation Influences Telomerase-Independent Telomere Maintenance Pathway Choice in .

G3 (Bethesda) 2018 07 31;8(8):2551-2558. Epub 2018 Jul 31.

European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, the Netherlands

In 10-15% of cancers, telomere length is maintained by a telomerase-independent, recombination-mediated pathway called alternative lengthening of telomeres (ALT). ALT mechanisms were first seen, and have been best studied, in telomerase-null cells called "survivors". There are two main types of survivors. Type I survivors amplify Y' subtelomeric elements while type II survivors, similar to the majority of human ALT cells, amplify the terminal telomeric repeats. Both types of survivors require Rad52, a key homologous recombination protein, and Pol32, a non-essential subunit of DNA polymerase δ. A number of additional proteins have been reported to be important for either type I or type II survivor formation, but it is still unclear how these two pathways maintain telomeres. In this study, we performed a genome-wide screen to identify novel genes that are important for the formation of type II ALT-like survivors. We identified 23 genes that disrupt type II survivor formation when deleted. 17 of these genes had not been previously reported to do so. Several of these genes (, , and ) are known to be involved in the regulation of dNTP levels. We find that dNTP levels are elevated early after telomerase inactivation and that this increase favors the formation of type II survivors.
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http://dx.doi.org/10.1534/g3.118.200280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071591PMC
July 2018

SAMHD1 acts at stalled replication forks to prevent interferon induction.

Nature 2018 05 18;557(7703):57-61. Epub 2018 Apr 18.

Institut de Génétique Humaine, CNRS, Université de Montpellier, Laboratoire Maintien de l'Intégrité du Génome au cours de la Réplication, Ligue Contre le Cancer, Montpellier, France.

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.
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http://dx.doi.org/10.1038/s41586-018-0050-1DOI Listing
May 2018

Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

Nucleic Acids Res 2018 06;46(11):e66

Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.

Information about the intracellular concentration of dNTPs and NTPs is important for studies of the mechanisms of DNA replication and repair, but the low concentration of dNTPs and their chemical similarity to NTPs present a challenge for their measurement. Here, we describe a new rapid and sensitive method utilizing hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the simultaneous determination of dNTPs and NTPs in biological samples. The developed method showed linearity (R2 > 0.99) in wide concentration ranges and could accurately quantify dNTPs and NTPs at low pmol levels. The intra-day and inter-day precision were below 13%, and the relative recovery was between 92% and 108%. In comparison with other chromatographic methods, the current method has shorter analysis times and simpler sample pre-treatment steps, and it utilizes an ion-pair-free mobile phase that enhances mass-spectrometric detection. Using this method, we determined dNTP and NTP concentrations in actively dividing and quiescent mouse fibroblasts.
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http://dx.doi.org/10.1093/nar/gky203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009580PMC
June 2018

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

PLoS Genet 2018 01 23;14(1):e1007129. Epub 2018 Jan 23.

Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5779651PMC
January 2018

Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired.

Proc Natl Acad Sci U S A 2017 11 6;114(47):12466-12471. Epub 2017 Nov 6.

Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden;

Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.
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http://dx.doi.org/10.1073/pnas.1713085114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703314PMC
November 2017

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

PLoS Genet 2017 Oct 25;13(10):e1007082. Epub 2017 Oct 25.

Dept. of Molecular Microbiology & Biotechnology, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673236PMC
October 2017

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress.

Mol Cell 2017 Oct 12;68(2):446-455.e3. Epub 2017 Oct 12.

Institute for Cancer Genetics and Department of Pediatrics and Genetics and Development, Columbia University, New York, NY 10032. Electronic address:

The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.
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http://dx.doi.org/10.1016/j.molcel.2017.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802358PMC
October 2017

Alterations in cellular metabolism triggered by or inactivation cause imbalanced dNTP pools and increased mutagenesis.

Proc Natl Acad Sci U S A 2017 05 17;114(22):E4442-E4451. Epub 2017 Apr 17.

German Cancer Research Center, 69120 Heidelberg, Germany;

Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility. To identify genes not previously linked to high-fidelity DNA replication, we conducted a genome-wide screen in using DNA polymerase active-site mutants as a "sensitized mutator background." Among the genes identified in our screen, three metabolism-related genes (, , and ) have not been previously associated to the suppression of mutations. Loss of either the transcription factor Gln3 or inactivation of the CTP synthetase Ura7 both resulted in the activation of the DNA damage response and imbalanced dNTP pools. Importantly, these dNTP imbalances are strongly mutagenic in genetic backgrounds where DNA polymerase function or MMR activity is partially compromised. Previous reports have shown that dNTP pool imbalances can be caused by mutations altering the allosteric regulation of enzymes involved in dNTP biosynthesis (e.g., RNR or dCMP deaminase). Here, we provide evidence that mutations affecting genes involved in RNR substrate production can cause dNTP imbalances, which cannot be compensated by RNR or other enzymatic activities. Moreover, Gln3 inactivation links nutrient deprivation to increased mutagenesis. Our results suggest that similar genetic interactions could drive mutator phenotypes in cancer cells.
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http://dx.doi.org/10.1073/pnas.1618714114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465912PMC
May 2017

Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations.

Nucleic Acids Res 2017 02;45(3):1200-1218

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.

In yeast, dNTP pools expand drastically during DNA damage response. We show that similar dNTP elevation occurs in strains, in which intrinsic replisome defects promote the participation of error-prone DNA polymerase ζ (Polζ) in replication of undamaged DNA. To understand the significance of dNTP pools increase for Polζ function, we studied the activity and fidelity of four-subunit Polζ (Polζ4) and Polζ4-Rev1 (Polζ5) complexes in vitro at ‘normal S-phase’ and ‘damage-response’ dNTP concentrations. The presence of Rev1 inhibited the activity of Polζ and greatly increased the rate of all three ‘X-dCTP’ mispairs, which Polζ4 alone made extremely inefficiently. Both Polζ4 and Polζ5 were most promiscuous at G nucleotides and frequently generated multiple closely spaced sequence changes. Surprisingly, the shift from ‘S-phase’ to ‘damage-response’ dNTP levels only minimally affected the activity, fidelity and error specificity of Polζ complexes. Moreover, Polζ-dependent mutagenesis triggered by replisome defects or UV irradiation in vivo was not decreased when dNTP synthesis was suppressed by hydroxyurea, indicating that Polζ function does not require high dNTP levels. The results support a model wherein dNTP elevation is needed to facilitate non-mutagenic tolerance pathways, while Polζ synthesis represents a unique mechanism of rescuing stalled replication when dNTP supply is low.
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http://dx.doi.org/10.1093/nar/gkw1149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388397PMC
February 2017

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

Cell Rep 2016 11;17(6):1657-1670

Department of Molecular and Cellular Biochemistry, Indiana University, Simon Hall MSB, 212 South Hawthorne Drive, Bloomington, IN 47405, USA; Department of Biology, Indiana University, Simon Hall MSB, 212 South Hawthorne Drive, Bloomington, IN 47405, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2016.10.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5134839PMC
November 2016

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

Nucleic Acids Res 2016 05 16;44(9):4222-32. Epub 2016 Apr 16.

Department of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

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.
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http://dx.doi.org/10.1093/nar/gkw268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872117PMC
May 2016

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

Proc Natl Acad Sci U S A 2016 Apr 11;113(17):4723-8. Epub 2016 Apr 11.

Department of Medical Biochemistry and Biophysics, Umeå University, Umea SE 901 87, Sweden;

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.
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http://dx.doi.org/10.1073/pnas.1519128113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855590PMC
April 2016
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