DOCSLIB.ORG

Genetic, Structural, and Functional Characterization of POLE Polymerase Proofreading Variants Allows Cancer Risk Prediction

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Genetic, Structural, and Functional Characterization of POLE Polymerase Proofreading Variants Allows Cancer Risk Prediction © American College of Medical Genetics and Genomics ARTICLE Genetic, structural, and functional characterization of POLE polymerase proofreading variants allows cancer risk prediction Nadim Hamzaoui, MD, PhD 1, Flora Alarcon, PhD2, Nicolas Leulliot, PhD3, Rosine Guimbaud, MD, PhD4, Bruno Buecher, MD, PhD5, Chrystelle Colas, MD, PhD6, Carole Corsini, MD7, Gregory Nuel, PhD8, Benoît Terris, MD, PhD9, Pierre Laurent-Puig, MD, PhD10, Stanislas Chaussade, MD11, Marion Dhooge, MD11, Clément Madru, PhD3 and Eric Clauser, MD, PhD1,12 Purpose: Polymerase proofreading-associated polyposis is a found in ten families. The estimated cumulative risk of CRC at 30, dominantly inherited colorectal cancer syndrome caused by 50, and 70 years was 11.1% (95% confidence interval [CI]: exonuclease domain missense variants in the DNA polymerases 4.2–17.5), 48.5% (33.2–60.3), and 74% (51.6–86.1). Cumulative POLE and POLD1. Manifestations may also include malignancies at risk of glioblastoma was 18.7% (3.2–25.8) at 70 years. Variants extracolonic sites. Cancer risks in this syndrome are not yet interfering with DNA binding (P286L and N363K) had a accurately quantified. significantly higher mutagenic effect than variants disrupting ion Methods: We sequenced POLE and POLD1 exonuclease domains metal coordination at the exonuclease site. in 354 individuals with early/familial colorectal cancer (CRC) or Conclusion: The risk estimates derived from this study provide a adenomatous polyposis. We assessed the pathogenicity of POLE rational basis on which to provide genetic counseling to POLE variants with yeast fluctuation assays and structural modeling. We variant carriers. estimated the penetrance function for each cancer site in variant carriers with a previously published nonparametric method Genetics in Medicine (2020) 22:1533–1541; https://doi.org/10.1038/s41436- based on survival analysis approach, able to manage unknown 020-0828-z genotypes. Results: Pathogenic POLE exonuclease domain variants P286L, Keywords: colorectal cancer; glioblastoma; penetrance; POLE; M294R, P324L, N363K, D368N, L424V, K425R, and P436S were polymerase proofreading-associated polyposis INTRODUCTION often show an “ultramutator” phenotype characterized by – Polymerase proofreading-associated polyposis (PPAP; OMIM mutational loads exceeding 100 variants per megabase2 5. 61259 and 615083), is an autosomal dominant cancer Knowledge of the age-specific cumulative cancer risks syndrome caused by the inability of the main replicative associated with PPAP could lead to improved cancer screen- DNA polymerases, POLE and POLD1, to proofread newly ing strategies and risk-reducing treatments. So far, only synthesized DNA strands1. Individuals with PPAP carry one publication has attempted to give colorectal cancer germline variants of the 3´–5´ proofreading exonuclease penetrance estimates in PPAP6. In the present study, we domain of POLE or POLD1 and are at increased risk of analyze the structural and functional consequences of eight colorectal cancer and adenoma. Several other tumor types, different POLE exonuclease variants found in ten new PPAP including endometrial, ovarian, duodenal, and brain cancers, families and give new estimates of the cumulative cancer risks have also been described in these families2. These tumors associated with PPAP. 1Service de Génétique et Biologie Moléculaires, Hôpital Cochin, APHP Centre Université de Paris and Inserm UMR_S1016, Institut Cochin, Université de Paris, Paris, France; 2Mathématiques appliquées Paris 5 (MAP5) CNRS: UMR8145, Université de Paris, Paris, France; 3Laboratoire de Cristallographie et RMN Biologiques, UMR CNRS 8015, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie de Paris, Paris, France; 4Unité d’oncogénétique, Institut Claudius Regaud, IUCT-Oncopole, Toulouse, France; 5Department of Medical Oncology, Institut Curie, Paris Sciences Lettres Research University, Paris, France; 6Department of Genetics, Institut Curie, Paris Sciences Lettres Research University, Paris, France; 7Medical Genetics Department, Centre Hospitalier Regional Universitaire de Montpellier, Montpellier, Languedoc-Roussillon, France; 8Institute of Mathematics, National Center for French Research, Laboratory of Probability, University Pierre et Marie Curie, Sorbonne University, Paris, France; 9Department of Pathology, Cochin Hospital, Assistance Publique–Hôpitaux de Paris, Paris, France; 10Department of Biochemistry, Unit of Pharmacogenetics and Molecular Oncology, Georges Pompidou European Hospital, Assistance Publique–Hôpitaux de Paris, Paris, France; 11Department of gastroenterology, Cochin hospital, assistance publique–Hôpitaux de Paris, Paris Descartes university, Paris, France; 12INSERM U970, Paris Descartes University, Paris, France. Correspondence: Nadim Hamzaoui (nadim. [email protected]) Submitted 4 December 2019; revised 27 April 2020; accepted: 27 April 2020 Published online: 19 May 2020 GENETICS in MEDICINE | Volume 22 | Number 9 | September 2020 1533 ARTICLE HAMZAOUI et al MATERIALS AND METHODS is taken into account as described12. The method has been Patients described in detail elsewhere13. Analysis was performed by We recruited 354 consecutive patients exhibiting features of using R software. In the present study, the covariate tested was early-onset/familial colorectal cancer (CRC) (218 individuals) gender and a difference between groups was tested using a and/or attenuated adenomatous polyposis (136 individuals) likelihood ratio test. between 2014 and 2017. Early-onset/familial CRC patients met the revised Bethesda criteria for microsatellite instability Strains and plasmid construction (MSI) testing7. Multiple colonic adenomas patients met the The Saccharomyces cerevisiae (MATa ade5-1 lys2-InsEA14 French National Cancer Institute criteria for MUTYH trp1-289 his7-2 leu2-3,112 ura3-52) yeast strain used in the screening8. We also included in this group patients for whom study is isogenic to E13414. Nucleotides 372–6669 of POL2 endoscopy and pathology reports gave descriptions like (the yeast homolog of human POLE) were amplified, cloned “polyposis” or “multiple polyps” rather than the exact number in pGEMT (Promega) and assembled in the pFA6a-KanMX6 of adenomas. A group of 253 patients were analyzed for the integration plasmid by restriction enzyme cloning. Exonu- first time, whereas 101 patients had been previously tested clease domain variants Pol2-P301L, Pol2-M309R, Pol2- negative for Lynch syndrome or adenomatous polyposis at P339L, Pol2-N378K, Pol2-D383N, Pol2-L439V, Pol2-K440R, other institutions. Written informed consent was obtained Pol2-A445T, and Pol2-P451S (encoding variants equivalent to from all subjects and the study received the approval of the human POLE P286L, M294R, P324L, N363K, D368N, L424V, Ethical Review Committee of the Cochin University Hospital. K425R, A430T, and P436S) were created with the Quik- Change Lightning Site-Directed Mutagenesis Kit (Agilent). Variant detection Plasmids were integrated into the POL2 locus after lineariza- A custom-made Ion Torrent Ion AmpliSeq panel (Thermo tion with Bsu36I and selection for G418 resistance, following 1234567890():,; Fisher Scientific, Waltham, MA) and a Ion PGM system the protocol described by Gietz and Schiestl15. Constructs (Thermo Fisher) were used to sequence the entire coding were verified by sequencing. Two control strains were also regions of 11 genes associated with hereditary digestive constructed using the same procedure, a negative control cancers (APC, MUTYH, AXIN2, MLH1, MSH2, MSH6, Pol2-wt (wild-type) strain and a positive control Pol2-AIA CDH1, STK11, PTEN, SMAD4, BMPR1A) and the exons strain defective in 3’–5’ proofreading exonuclease by variants coding the exonuclease domains of POLE and POLD1 of the highly conserved Exo I motif DIE into an AIA motif (codons 268–471 of POLE and 304–517 of POLD1). A search (residues 290–292). for large rearrangements in the genes APC, MLH1, MSH2, MSH6, and EPCAM was done by multiplex ligation- Fluctuation assays dependent probe amplification (MLPA) (MRC Holland). The spontaneous variant rate was measured by fluctuation Cases with loss of MLH1 protein expression in the tumor by analysis according to Lang and Murray16. For mutant immunohistochemistry and without germline MLH1 deleter- selection, yeast cultures were plated onto two types of ious variants were further studied by methylation-specific selective media: CAN10X (synthetic complete medium with- MLPA (MRC Holland) searching for constitutional epimuta- out arginine, 0.5 g/liter of L-canavanine) and HIS- (synthetic tion of MLH1. complete medium without histidine). Canavanine is a toxic arginine analog, whose uptake requires the arginine trans- Estimation of penetrance porter encoded by the CAN1 gene. The Canr forward variant The objective was to estimate the penetrance function in the assay selects for loss-of-function mutants of this transporter, context where most genotypes are unobserved. For this which occur through a variety of base substitutions, frame- purpose, we use a nonparametric method based on a survival shifts, and larger rearrangements. The his7-2 reporter is a -1 analysis approach. Since many genotypes are unobserved, we frameshift variant creating a run of 7 adenines. Gain-of- + use a classical expectation–maximization (EM) algorithm9 to function mutants selected by the His reverse variant assay estimate iteratively both the penetrance function and the occur primarily by +1 or -2 indel changes. All experiments posterior carrier probability.
Load more

Recommended publications
  • DNA Proofreading and Repair
    DNA proofreading and repair Mechanisms to correct errors during DNA replication and to repair DNA damage over the cell's lifetime. Key points: Cells have a variety of mechanisms to prevent mutations, or permanent changes in DNA sequence. During DNA synthesis, most DNA polymerases "check their work," fixing the majority of mispaired bases in a process called proofreading. Immediately after DNA synthesis, any remaining mispaired bases can be detected and replaced in a process called mismatch repair. If DNA gets damaged, it can be repaired by various mechanisms, including chemical reversal, excision repair, and double-stranded break repair. Introduction What does DNA have to do with cancer? Cancer occurs when cells divide in an uncontrolled way, ignoring normal "stop" signals and producing a tumor. This bad behavior is caused by accumulated mutations, or permanent sequence changes in the cells' DNA. Replication errors and DNA damage are actually happening in the cells of our bodies all the time. In most cases, however, they don’t cause cancer, or even mutations. That’s because they are usually detected and fixed by DNA proofreading and repair mechanisms. Or, if the damage cannot be fixed, the cell will undergo programmed cell death (apoptosis) to avoid passing on the faulty DNA. Mutations happen, and get passed on to daughter cells, only when these mechanisms fail. Cancer, in turn, develops only when multiple mutations in division-related genes accumulate in the same cell. In this article, we’ll take a closer look at the mechanisms used by cells to correct replication errors and fix DNA damage, including: Proofreading, which corrects errors during DNA replication Mismatch repair, which fixes mispaired bases right after DNA replication DNA damage repair pathways, which detect and correct damage throughout the cell cycle Proofreading DNA polymerases are the enzymes that build DNA in cells.
    [Show full text]
  • Two Proofreading Steps Amplify the Accuracy of Genetic Code Translation
    Two proofreading steps amplify the accuracy of genetic code translation Ka-Weng Ieonga, Ülkü Uzuna,1, Maria Selmera, and Måns Ehrenberga,2 aDepartment of Cell and Molecular Biology, Uppsala University, Uppsala 75124, Sweden Edited by Ada Yonath, Weizmann Institute of Science, Rehovot, Israel, and approved October 12, 2016 (received for review July 4, 2016) Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA kinetic efficiency to substrate-selective, enzyme-catalyzed reactions programmed ribosome in ternary complex with elongation factor than single-step proofreading (5, 14, 15), it has been taken for Tu (EF-Tu) and GTP and then, again, in a proofreading step after granted that there is but a single proofreading step in tRNA se- GTP hydrolysis on EF-Tu. We use tRNA mutants with different lection by the translating ribosome (16). Here, we present data affinities for EF-Tu to demonstrate that proofreading of aa- showing that the proofreading factor (F), by which the accuracy (A) tRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary is amplified from its initial selection value (I) by aa-tRNA in ternary complex with EF-Tu·GDP are selected in a step where the accu- complex with EF-Tu and GTP, increases linearly with increasing racy increases linearly with increasing aa-tRNA affinity to EF-Tu. association equilibrium constant, KA, for aa-tRNA binding to EF- Then, following dissociation of EF-Tu·GDP from the ribosome, the Tu. We suggest the cause of this linear increase to be the activity of accuracy is further increased in a second and apparently EF- a first proofreading step, in which aa-tRNA is discarded in complex Tu−independent step.
    [Show full text]
  • Proofreading-Defective DNA Polymerase II Increases Adaptive
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7951-7955, August 1995 Biochemistry Proofreading-defective DNA polymerase II increases adaptive mutation in Escherichia coli (polB gene/3' exonuclease/mismatch repair/DNA polymerase III/antimutator) PATRICIA L. FOSTER*, GUDMUNDUR GUDMUNDSSONt, JEFFREY M. TRIMARCHI*, HONG CAIt, AND MYRON F. GOODMANtt *Department of EnvironmentalHealth, Boston University School of Public Health, Boston, MA 02118-2394; and tDepartment of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, CA 90089-1340 Communicated by Evelyn M. Witkin, Rutgers, The State University of New Jersey, Piscataway, NJ, June 5, 1995 ABSTRACT The role ofEscherichia coli DNA polymerase strain, FC40, its F- parent, FC36, the scavenger F' AlaclZ (Pol) II in producing or avoiding mutations was investigated strain, FC29, and thepolBAl derivative of FC40, FCB60, have by replacing the chromosomal Pol II gene (polB+) by a gene been described (10, 12, 13). ApolBexl derivative of FC40 was encoding an exonuclease-deficient mutant Pol II (polBexi). made from HC203 by transduction to arabinose utilization. The polBexi allele increased adaptive mutations on an epi- The dnaE915 or control dnaE+ strains were made from some in nondividing cells under lactose selection. The pres- NR9915 or NR9918 by transducing to tetracycline resistance ence of a Pol III antimutator allele (dnaE915) reduced adap- and then screening for chloramphenicol resistance. In all cases tive mutations in both polB+ cells and cells deleted for polB the F' 1ac133::1acZ was mated into the strain last, and then the (polBAl) to below the wild-type level, suggesting that both Pol phenotypes of several independent isolates were tested.
    [Show full text]
  • A Dinb Variant Reveals Diverse Physiological Consequences of Incomplete TLS Extension by a Y-Family DNA Polymerase
    A DinB variant reveals diverse physiological consequences of incomplete TLS extension by a Y-family DNA polymerase The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Jarosz, D. F. et al. “A DinB variant reveals diverse physiological consequences of incomplete TLS extension by a Y-family DNA polymerase.” Proceedings of the National Academy of Sciences 106.50 (2009): 21137-21142. Copyright ©2011 by the National Academy of Sciences As Published http://dx.doi.org/10.1073/pnas.0907257106 Publisher National Academy of Sciences (U.S.) Version Final published version Citable link http://hdl.handle.net/1721.1/61367 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. A DinB variant reveals diverse physiological consequences of incomplete TLS extension by a Y-family DNA polymerase Daniel F. Jarosza,1, Susan E. Cohenb, James C. Delaneyc, John M. Essigmanna,c, and Graham C. Walkerb,2 Departments of aChemistry, bBiology, and cBiological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved October 20, 2009 (received for review June 30, 2009) The only Y-family DNA polymerase conserved among all domains however. The active sites of both pol ␬ (41) and DinB (22) are of life, DinB and its mammalian ortholog pol ␬, catalyzes proficient somewhat closed under many conditions. This may occur at least in bypass of damaged DNA in translesion synthesis (TLS).
    [Show full text]
  • Microevolution of Candida Albicans Isolate from a Patient with Mucocutaneous Candidiasis and HIV Infection
    Open Journal of Medical Microbiology, 2017, 7, 41-49 http://www.scirp.org/journal/ojmm ISSN Online: 2165-3380 ISSN Print: 2165-3372 Microevolution of Candida albicans Isolate from a Patient with Mucocutaneous Candidiasis and HIV Infection Gabriel Palma Cortés1, Carlos Cabello Gutierrez1, Misael González Ibarra2, Magdalena Aguirre García3, Fernando Hernández Sánchez1, Haydee Torres Guerrero3* 1Departamento de Investigación en Virología y Micología, Instituto Nacional de Enfermedades Respiratoria “Ismael Cosío Villegas”, Ciudad de México, México 2Laboratorio de Inmuno Alergología y Micología Médica, División de Investigación, Hospital Juárez de México, Ciudad de México, México 3Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, Hospital General de México “Dr. Eduardo Liceaga”, Ciudad de México, México How to cite this paper: Palma Cortés, G., Abstract Gutierrez, C.C., Ibarra, M.G., García, M.A., Sánchez, F.H. and Guerrero, H.T. (2017) Candidiasis is the most common opportunistic fungal infection in HIV pa- Microevolution of Candida albicans Isolate tients, and its presence is ascribed mainly to the persistence of the original in- from a Patient with Mucocutaneous Can- fecting strain. The latter might acquire genetic variations during interaction didiasis and HIV Infection. Open Journal of Medical Microbiology, 7, 41-49. with the host, reflecting the adaptation of the strain. Here, we report the case https://doi.org/10.4236/ojmm.2017.72004 of a 32-year-old man complaining of asthenia, irregular hyperpyrexia, and dry cough, who was admitted to the emergency unit. Laboratory examination Received: March 7, 2017 Accepted: June 17, 2017 showed positivity for HIV. Dark violet macular lesions and ulcerated lesions Published: June 20, 2017 with verrucous erosion were observed at the tip of the nose, whereas an ulcer without exudates was noted in the pubic region.
    [Show full text]
  • Lecture 25: DNA Mutation, Proofreading, and Repair
    Lecture 25: DNA mutation, Lecture Outline 11/2/05 proofreading, and repair • Review DNA replication machine G • Fidelity of replication and proofreading C A T T A • Replicating the ends of chromosomes 1 nm G C 3.4 nm C G • Mutation A T C G – Types of mutations T A T A – Repair mechanisms A T A T G C 0.34 nm A T Figure 16.7a, c (c) Space-filling model 1 2 Replication overview DNA Polymerase III • Look at animations on your textbook CD • A complex enzyme with many subunits • one part adds the nucleotides • another helps it slide along the template • Look again at the animation from DNAi • another checks for mis-pairing – http://www.dnai.org – (go to the section on copying the code) 3 4 Figs. from http://www.mun.ca/biochem/courses/3107 1 Proofreading Fidelity of replication • Even though bases preferentially pair G-C and A-T, Replication step error rate the initial error rate is about 1 in 10,000. 5䈊䊲㻖䈊㻃polymerization 1 × 105 • Many polymerases have “proofreading” ability. They 3䈊䊲㻘䈊 proofreading 1 × 102 can excise an mis-paired base and try again. Strand-directed mismatch repair 1 × 102 • This reduces the error rate to about 1 in a billion. One polymerase subunit adds nucleotides Total error rate 1 × 109 Another “edits” out incorrec5t bases 6 What happens to the lagging strand The ends of eukaryotic chromosomal DNA get at the end of the chromosome? shorter with each round of replication 5′ End of parental Leading strand DNA strands Lagging strand 3′ Leaves a gap when the RNA Last fragment Previous fragment primer is removed RNA primer 5′
    [Show full text]
  • Original Research Article an Overview of the Proofreading Functions In
    1 Original Research Article An Overview of the Proofreading Functions in Bacteria and in Severe Acute Respiratory Syndrome- Coronaviruses ABSTRACT Aim: To understand the structure-function relationship of the proofreading (PR) functions in eubacteria and viruses with special reference to Severe Acute Respiratory Syndrome-Coronaviruses (SARS-CoVs) and propose a plausible mechanism of action for PR exonucleases of SARS-CoVs. Study Design: Bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallographic data were used to study the structure-function relationships of the PR exonucleases from bacteria and CoVs. Methodology: The protein sequences of the PR exonucleases of various DNA polymerases, and RNA polymerases of SARS, SARS-related and human CoVs (HCoVs) were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical, SDM experiments and X-ray crystallographic analysis on these enzymes were used to arrive at the possible active amino acids in the PR exonucleases of these crucial enzymes. Results: A complete analysis of the active sites of the PR exonucleases from various bacteria and CoVs were done. The multiple sequence alignment (MSA) analysis showed many conserved amino acids, small and large peptide regions among them. Based on the conserved motifs, the PR exonucleases are found to fit broadly into two superfamilies, viz. DEDD and polymerase-histidinol phosphatase (PHP) superfamilies. The bacterial DNA polymerases I and II, RNase D, RNase T and ε-subunit of DNA polymerases III belong to the DEDD superfamily. The PR enzymes from SARS, SARS-related CoVs and other HCoVs also essentially belong to the DEDD superfamily.
    [Show full text]
  • Error-Prone Bypass of Certain DNA Lesions by the Human DNA Polymerase Κ
    Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION mologies with UmuC, whereas Rev3, the catalytic sub- Error-prone bypass of certain unit of pol ␨ is similar to replicative DNA polymerases. DNA lesions by the human Recently, a human homolog of Rev3 was identified and DNA polymerase ␬ shown to be involved in UV-induced mutagenesis (Gibbs et al. 1998). Furthermore, the gene responsible for the Eiji Ohashi, Tomoo Ogi, Rika Kusumoto,1 human disease xeroderma pigmentosum variant (XP-V) Shigenori Iwai,2 Chikahide Masutani,1 was shown to code for a Rad30 homolog that bypasses a Fumio Hanaoka,1 and Haruo Ohmori3 cys–syn T-T dimer, but not a (6-4) T-T dimer (Johnson et al. 1999a; Masutani et al. 1999a,b). The S. cerevisiae Institute for Virus Research, Kyoto University, Kyoto, Kyoto Rad30 and the human XPV enzymes are considered to be 606-8507, Japan; 1Institute for Molecular and Cellular Biology, error-free because they insert two dAMPs opposite a cys– Osaka University, and CREST, Japan Science and Technology syn T-T dimer in vitro and the absence of their activities 2 Corporation, Suita, Osaka 565-0871, Japan; and Biomolecular leads to enhanced levels of mutagenesis in vivo (Johnson Engineering Research Institute, Suita, Osaka 565-0874, Japan et al. 1999a,b,c; Masutani et al. 1999a,b). The Escherichia coli protein DinB is a newly identified E. coli has another DNA polymerase (DinB, pol IV) error-prone DNA polymerase. Recently, a human homo- (Wagner et al. 1999) with some similarity in amino-acid log of DinB was identified and named DINB1.
    [Show full text]
  • Rapid Microevolution of Biofilm Cells in Response to Antibiotics
    www.nature.com/npjbiofilms ARTICLE OPEN Rapid microevolution of biofilm cells in response to antibiotics Anahit Penesyan 1,2, Stephanie S. Nagy 1, Staffan Kjelleberg3,4,5, Michael R. Gillings 6 and Ian T. Paulsen1* Infections caused by Acinetobacter baumannii are increasingly antibiotic resistant, generating a significant public health problem. Like many bacteria, A. baumannii adopts a biofilm lifestyle that enhances its antibiotic resistance and environmental resilience. Biofilms represent the predominant mode of microbial life, but research into antibiotic resistance has mainly focused on planktonic cells. We investigated the dynamics of A. baumannii biofilms in the presence of antibiotics. A 3-day exposure of A. baumannii biofilms to sub-inhibitory concentrations of antibiotics had a profound effect, increasing biofilm formation and antibiotic resistance in the majority of biofilm dispersal isolates. Cells dispersing from biofilms were genome sequenced to identify mutations accumulating in their genomes, and network analysis linked these mutations to their phenotypes. Transcriptomics of biofilms confirmed the network analysis results, revealing novel gene functions of relevance to both resistance and biofilm formation. This approach is a rapid and objective tool for investigating resistance dynamics of biofilms. npj Biofilms and Microbiomes (2019) 5:34 ; https://doi.org/10.1038/s41522-019-0108-3 INTRODUCTION as “biofilm tolerance”15–17) via biofilm-specific mechanisms such Acinetobacter baumannii is a Gram-negative pathogen found in as the shielding effect of the biofilm matrix that leads to restricted 1234567890():,; 18 hospitals worldwide.1 It is responsible for opportunistic infections penetration of antimicrobials, the slower growth rate in deep 19 20 of the bloodstream, urinary tract, and other soft tissues, and can layers of biofilms, and the presence of persister cells.
    [Show full text]
  • Formation and Recognition of UV-Induced DNA Damage Within Genome Complexity
    International Journal of Molecular Sciences Review Formation and Recognition of UV-Induced DNA Damage within Genome Complexity Philippe Johann to Berens and Jean Molinier * Institut de Biologie Moléculaire des Plantes du CNRS, 12 rue du Général Zimmer, 67000 Strasbourg, France; [email protected] * Correspondence: [email protected] Received: 31 July 2020; Accepted: 9 September 2020; Published: 12 September 2020 Abstract: Ultraviolet (UV) light is a natural genotoxic agent leading to the formation of photolesions endangering the genomic integrity and thereby the survival of living organisms. To prevent the mutagenetic effect of UV, several specific DNA repair mechanisms are mobilized to accurately maintain genome integrity at photodamaged sites within the complexity of genome structures. However, a fundamental gap remains to be filled in the identification and characterization of factors at the nexus of UV-induced DNA damage, DNA repair, and epigenetics. This review brings together the impact of the epigenomic context on the susceptibility of genomic regions to form photodamage and focuses on the mechanisms of photolesions recognition through the different DNA repair pathways. Keywords: ultraviolet; photolesions; photodamage recognition; chromatin; photolyase; nucleotide excision repair; transcription coupled repair; global genome repair 1. Introduction Solar radiation that reaches the surface of the Earth consists of 3 main spectra: ultraviolet (UV; 100–400 nm), visible light (400–700 nm), and infrared (IR; 700 nm to over 1 mm). Each of these ranges of wavelengths plays essential roles in providing light, heat, and energy, allowing the proper development of life. In addition to their beneficial impacts for living organisms, these types of irradiation can lead to deleterious effects, affecting cellular structures, interfering with biological processes, and damaging DNA.
    [Show full text]
  • Proofreading and Inviability Due to Error Catastrophe
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2856-2861, April 1996 Genetics Mutants in the Exo I motif of Escherichia coli dnaQ: Defective proofreading and inviability due to error catastrophe (fidelity of DNA replication/dnaQ and dnaE genes/mutD5 mutator/dnaE antimutators/saturation of mismatch repair) IWONA J. FIJALKOWSKA* AND ROEL M. SCHAAPERt Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 Communicated by Maurice S. Fox, Massachusetts Institute of Technology, Cambridge, MA, December 11, 1995 (received for review October 27, 1995) ABSTRACT The Escherichia coli dnaQ gene encodes the subunit: its exonuclease function and its presumed structural proofreading 3' exonuclease (e subunit) of DNA polymerase role within the replication complex based on its tight binding III holoenzyme and is a critical determinant of chromosomal to both the a and 0 subunits (2). An understanding of the replication fidelity. We constructed by site-specific mutagen- specific role of the (proofreading) exonuclease may thus esis a mutant, dnaQ926, by changing two conserved amino require a mutant that is devoid of exonuclease activity but acid residues (Asp-12-Ala and Glu-14--Ala) in the Exo I otherwise normal in its protein-protein interactions. One motif, which, by analogy to other proofreading exonucleases, possible candidate is the mutD5 mutant (8, 9), which carries a is essential for the catalytic activity. When residing on a mutation in dnaQ (10, 11). mutD5 is an exceptionally strong plasmid, dnaQ926 confers a strong, dominant mutator phe- mutator, mutation rates being enhanced 104- to 105-fold in rich notype, suggesting that the protein, although deficient in medium (8, 9, 12).
    [Show full text]
  • Low Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly-Regulated Double-Edged Sword. Malgorzata Jaszczur, Jeffrey G Bertram, Andrew Robinson, Antoine M
    Subscriber access provided by UNIV OF WISCONSIN - MADISON Current Topic/Perspective Mutations for Worse or Better: Low Fidelity DNA Synthesis by SOS DNA Polymerase V is a Tightly-Regulated Double-Edged Sword. Malgorzata Jaszczur, Jeffrey G Bertram, Andrew Robinson, Antoine M. van Oijen, Roger Woodgate, Michael M. Cox, and Myron F Goodman Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00117 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 7, 2016 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain.
    [Show full text]