Publications by authors named "Sushma Sharma"

83 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

Indian classical music with incremental variation in tempo and octave promotes better anxiety reduction and controlled mind wandering-A randomised controlled EEG study.

Explore (NY) 2021 Mar-Apr;17(2):115-121. Epub 2020 Mar 11.

Kempegowda Institute of Medical Sciences (KIMS), Bengaluru, Karnataka, India.

Studies have reported the benefits of music-listening in stress-reduction using musical pieces of specific scale or 'Raaga'. But the influence of lower-level musical properties (like tempo, octave, timbre, etc.) lack research backing. Carnatic music concerts use incremental modulations in tempo and octave (e.g.: 'Ragam-Tanam-Pallavi') to elevate the mood of audiences. Therefore, the current study aimed to examine the anxiolytic effect of this musical property. A randomised controlled cross-over study with 21 male undergraduate medical students was followed. 11 participants listened to 'Varying music' (VM: instrumental music with incremental variations in tempo and octave) and 10 listened to 'Stable music' (SM: instrumental music without such variations), thrice daily for 6 days, both clips recorded in Raaga-Kaapi and silence being the control intervention. Electroencephalography (EEG) and Electrocardiography (for heart rate variability or HRV) were done on all 6 days. Beck's Anxiety inventory and State-trait anxiety scale were administered on Day-1 and Day-6. A significant anxiety score reduction was seen only in VM. VM showed marked decrease in lower frequency EEG power in bilateral temporo-parieto-occipital regions compared to silence, whereas SM showed increase in higher frequencies. Relatively, VM showed more midline power reduction (i.e., lower default mode network or DMN activity) and SM showed greater left-dominant alpha/beta asymmetry (i.e., greater right brain activation). During both music interventions HRV remained stable, unlike silence intervention. We speculate that, gradual transition between lower-slower and higher-faster music portions of VM induces a 'controlled-mind wandering' state involving balanced switching between heightened mind wandering ('attention to self') and reduced mind wandering ('attention to music') states, respectively. Therefore, music-selection has remarkable influence on stress-management and warrants further research.
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http://dx.doi.org/10.1016/j.explore.2020.02.013DOI Listing
March 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

Every dog has its prey: Range-wide assessment of links between diet patterns, livestock depredation and human interactions for an endangered carnivore.

Sci Total Environ 2020 Apr 18;714:136798. Epub 2020 Jan 18.

School of Natural Resources and Environment, University of Florida, Gainesville, USA; Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, USA.

Livestock depredation is the most ubiquitous type of negative interaction between humans and carnivores. We conducted a range-wide assessment linking diet patterns of the endangered dhole Cuon alpinus, with livestock consumption and human-dhole interactions. We first performed a reanalysis of dhole diet data from all published studies (1973-2013) incorporating a recently-developed non-linear correction factor for quantifying prey biomass consumed. We then determined the relative livestock numbers consumed by dholes over time across its range, compared these with earlier estimates, and investigated the relative importance of wild vs. non-wild prey in dhole diet. Using information from >70 studies, we explored links between livestock consumption by dholes, availability of wild versus non-wild prey, sympatric depredation-prone carnivores, and people's perception of dholes as livestock predators. We found that (a) dhole diet profiles varied regionally, (b) dholes consumed fewer livestock compared to estimates generated using other, widely used methods, (c) livestock consumption by dholes was associated with wild and non-wild prey densities, and number of co-predator species, and (d) people's negative perception of dholes was associated with pack sizes, levels of livestock depredation and number of sympatric carnivore species. Global efforts for dhole conservation should involve different strategies based on region-specific realities that account for ecological context as well as human perceptions, which would require well-designed studies of dhole social and population dynamics, and human-dhole interactions. We also call for more such range-wide assessments of livestock depredation by wild canids, complemented with direct investigations of human-canid interactions.
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http://dx.doi.org/10.1016/j.scitotenv.2020.136798DOI Listing
April 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

Five Best Practices for More Effective Use of Ambulatory Electronic Health Records to Manage Chronic Disease.

Tex Med 2019 Sep 1;115(9):e1. Epub 2019 Sep 1.

The prevalence of diabetes mellitus, which affects 30.3 million adults in the United States, is increasing with 1.5 million newly diagnosed cases of diabetes each year. An additional 84 million adults are affected by prediabetes. Consequently, this chronic disease has become the seventh leading cause of death in our nation.1 Statistics like these have caused the Centers for Medicare & Medicaid Services (CMS) and state health departments to encourage primary care physicians to not only adopt electronic health records (EHRs) but to use them more effectively to improve management of diabetes and other chronic diseases. This paper discusses how small physician practices in North Texas improved their tracking of quality metrics for hypertension, obesity, and diabetes by leveraging available but underused EHR functionalities. We also describe five "best practices" for more effective use of ambulatory EHRs to manage chronic disease based on findings in this study.
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September 2019

Primary care clinics can be a source of exposure to virulent Clostridium (now Clostridioides) difficile: An environmental screening study of hospitals and clinics in Dallas-Fort Worth region.

PLoS One 2019 15;14(8):e0220646. Epub 2019 Aug 15.

The Dallas-Fort Worth Hospital Council Education and Research Foundation, Irving, TX, United States of America.

C. difficile is an endospore-forming pathogen, which is becoming a common cause of microbial health-care associated gastrointestinal disease in the United States. Both healthy and symptomatic patients can shed C. difficile spores into the environment, which can survive for long periods, being resistant to desiccation, heat, and disinfectants. In healthcare facilities, environmental contamination with C. difficile is a major concern as a potential source of exposure to this pathogen and risk of disease in susceptible patients. Although hospital-acquired infection is recognized, community-acquired infection is an increasingly recognized health problem. Primary care clinics may be a significant source of exposure to this pathogen; however, there are limited data about presence of environmental C. difficile within clinics. To address the potential for primary care clinics as a source of environmental exposure to virulent C. difficile, we measured the frequency of environmental contamination with spores in clinic examination rooms and hospital rooms in Dallas-Fort Worth (DFW) area of Texas. The ribotypes and presence of toxin genes from some environmental isolates were compared. Our results indicate primary care clinics have higher frequencies of contamination than hospitals. After notification of the presence of C. difficile spores in the clinics and an educational discussion to emphasize the importance of this infection and methods of infection prevention, environmental contamination in clinics was reduced on subsequent sampling to that found in hospitals. Thus, primary care clinics can be a source of exposure to virulent C. difficile, and recognition of this possibility can result in improved infection prevention, potentially reducing community-acquired C. difficile infections and subsequent disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220646PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695158PMC
March 2020

Mck1 defines a key S-phase checkpoint effector in response to various degrees of replication threats.

PLoS Genet 2019 08 5;15(8):e1008136. Epub 2019 Aug 5.

State Key Laboratory of Agro-Biotechnology and Beijing Advanced Innovation Center for Food Nutrition and Human Health, MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, P.R. China.

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis.
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http://dx.doi.org/10.1371/journal.pgen.1008136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695201PMC
August 2019

Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress.

DNA Repair (Amst) 2019 11 24;83:102674. Epub 2019 Jul 24.

Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot, 76100, Israel. Electronic address:

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in dNTP biosynthesis and is tightly regulated at the transcription and activity levels. One of the best characterized responses of yeast to DNA damage is up-regulation of RNR transcription and activity and consequently, elevation of the dNTP pools. Hydroxyurea is a universal inhibitor of RNR that causes S phase arrest. It is used in the clinic to treat certain types of cancers. Here we studied the response of the fungal plant pathogen Fusarium oxysporum to hydroxyurea in order to generate hypotheses that can be used in the future in development of a new class of pesticides. F. oxysporum causes severe damage to more than 100 agricultural crops and specifically threatens banana cultivation world-wide. Although the recovery of F. oxysporum from transient hydroxyurea exposure was similar to the one of Saccharomyces cerevisiae, colony formation was strongly inhibited in F. oxysporum in comparison with S. cerevisiae. As expected, genomic and phosphoproteomic analyses of F. oxysporum conidia (spores) exposed to hydroxyurea showed hallmarks of DNA replication perturbation and activation of recombination. Unexpectedly and strikingly, RNR was not induced by either hydroxyurea or the DNA-damaging agent methyl methanesulfonate as determined at the RNA and protein levels. Consequently, dNTP concentrations were significantly reduced, even in response to a low dose of hydroxyurea. Methyl methanesulfonate treatment did not induce dNTP pools in F. oxysporum, in contrast to the response of RNR and dNTP pools to DNA damage and hydroxyurea in several tested organisms. Our results are important because the lack of a feedback mechanism to increase RNR expression in F. oxysporum is expected to sensitize the pathogen to a fungal-specific ribonucleotide inhibitor. The potential impact of our observations on F. oxysporum genome stability and genome evolution is discussed.
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http://dx.doi.org/10.1016/j.dnarep.2019.102674DOI Listing
November 2019

Possible Missed Acute Coronary Syndrome Rate in North Texas: Is There Room to Improve?

Crit Pathw Cardiol 2019 09;18(3):121-124

Department of Emergency Medicine, University of Texas at Southwestern, Dallas, TX.

Background: Acute coronary syndrome (ACS) is a common diagnosis in the emergency department (ED). Missing this diagnosis may lead to increased morbidity or mortality. With improved cardiac biomarkers tests, it is unknown if that has decreased the prevalence of ACS diagnoses in ED patients who were recently evaluated in the ED.

Methods: This is a retrospective review of ED patients who were diagnosed with ACS and seen in the ED 7 and 30 days before that visit in North Texas between 2009 and 2015. The demographics and temporal trends of missed ACS rates are described. Logistic regression was used to evaluate if any factors (ie, age, ethnicity, sex, insurance status) were significant.

Results: Between December 26, 2008 and June 29, 2015, there were 24,914 diagnoses of ACS in the ED. The overall prevalence of patients diagnosed with ACS 7 days after their ED visit was 3.2% and 8.8% at 30 days. For patients diagnosed with ACS 7 days and 30 days after an ED visit, the most common initial ED diagnoses were nonspecific chest pain (57.7%), atherosclerotic disease (19.5%), and heart failure (12.8%). Between 2009 and 2015, there was no overall change in the rate of ACS diagnoses in patients seen 7 or 30 days before an ED visit.

Conclusions: The prevalence of missed ACS in the North Texas region at 7 and 30 days after the initial ED visit is low and has not changed over the past several years.
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http://dx.doi.org/10.1097/HPC.0000000000000188DOI Listing
September 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

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

Trends in Geospatial Drivers of Fall-Related Hospitalizations and Asset Mapping of Fall Prevention Interventions for Vulnerable Older Adults.

J Aging Health 2020 Jun/Jul;32(5-6):328-339. Epub 2019 Jan 7.

Texas A&M University, College Station, USA.

Given that one in four older adults suffer potentially preventable falls annually, we aimed to identify areas with (a) delivery gaps of evidence-based programs (EBPs) targeting fall prevention among older adults, namely A Matter of Balance (AMOB), and (b) high rates of fall-related hospitalizations-hotspots. Analyses included multiple geospatially linked datasets. EBPs were delivered ≥1 time in 84 counties in 2012 and 90 counties in 2014. Factors associated with EPB delivery gaps (absence; <.05) included high-density older adult areas, non-fall-related hospitalization hotspots, lower population density, nonmetropolitan areas, high-density Hispanic adult areas, and areas with limited access to home health care agencies. Hotspots for fall-related hospitalization numbered 64 in 2012 and 62 in 2014. Factors associated with hotspots included low-density older adult areas, having AMOB delivered ≥1 time annually, high population density, and high-density Hispanic adult areas. In resource-finite settings (e.g., the aging services sector), identifying high priority areas allows for precise allocation of limited resources.
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http://dx.doi.org/10.1177/0898264318822381DOI Listing
January 2021

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

Trends in Chronic Liver Disease-Related Hospitalizations: A Population-Based Study.

Am J Gastroenterol 2019 01;114(1):98-106

Baylor College of Medicine, Houston, Texas, USA.

Objectives: In a population-based study, we examined time trends in chronic liver disease (CLD)-related hospitalizations in a large and diverse metroplex.

Methods: We examined all CLD-related inpatient encounters (2000-2015) in Dallas-Fort Worth (DFW) using data from the DFW council collaborative that captures claims data from 97% of all hospitalizations in DFW (10.7 million regional patients).

Results: There were 83,539 CLD-related hospitalizations in 48,580 unique patients across 84 hospitals. The age and gender standardized annual rate of CLD-related hospitalization increased from 48.9 per 100,000 in 2000 to 125.7 per 100,000 in 2014. Mean age at hospitalization increased from 54.0 (14.1) to 58.5 (13.5) years; the proportion of CLD patients above 65 years increased from 24.2% to 33.1%. HCV-related hospitalizations plateaued, whereas an increase was seen in hospitalizations related to alcohol (9.1 to 22.7 per 100,000) or fatty liver (1.4 per 100,000 to 19.5 per 100,000). The prevalence of medical comorbidities increased for CLD patients: coronary artery disease (4.8% to 14.3%), obesity (2.8% to 14.6%), chronic kidney disease (2.8% to 18.2%), and diabetes (18.0% to 33.2%). Overall hospitalizations with traditional complications of portal hypertension (ascites, varices, and peritonitis) remained stable over time. However, hospitalization with complications related to infection increased from 54.7% to 66.4%, and renal failure increased by sevenfold (2.7% to 19.5%).

Conclusions: CLD-related hospitalizations have increased twofold over the last decade. Hospitalized CLD patients are older and sicker with multiple chronic conditions. Traditional complications of portal hypertension have been superseded by infection and renal failure, warranting a need to redefine what it means to have decompensated CLD.
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http://dx.doi.org/10.1038/s41395-018-0365-4DOI Listing
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

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

Acacia gum polysaccharide based hydrogel wound dressings: Synthesis, characterization, drug delivery and biomedical properties.

Carbohydr Polym 2017 Jun 20;165:294-303. Epub 2017 Feb 20.

Department of Chemistry, Himachal Pradesh University, Shimla 171005, India.

Keeping in view the importance of polysaccharide gums for wound care, in the present article, an attempt has been made to explore antioxidant nature of gum acacia in designing hydrogel wound dressing to improve its wound healing potential. These polymers were prepared by using acacia gum-polyvinylpyrollidone/carbopol and were characterized by C NMR, FTIR, SEM, AFM, cryo-SEM, XRD, TGA, DSC and elemental analysis techniques. Some important biomaterial properties of wound dressings such as wound fluid absorption, haemo-compatibility, bioactive assessment, gaseous/water/microbial permeability, mechanical properties, bio-adhesion, drug release, and histology of wound healing were also determined. Hydrogel wound dressings were found non-haemolytic, antioxidant and mucoadhesive in nature. Release of drug occurred through non-Fickian diffusion mechanism and release profile best fitted in Higuchi model.
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http://dx.doi.org/10.1016/j.carbpol.2017.02.039DOI Listing
June 2017
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