DOCSLIB.ORG

Complex Modifier Landscape Underlying Genetic Background Effects

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Complex Modifier Landscape Underlying Genetic Background Effects Complex modifier landscape underlying genetic background effects Jing Houa,1, Guihong Tana, Gerald R. Finkb, Brenda J. Andrewsa,1, and Charles Boonea,1 aDonnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada; and bWhitehead Institute for Biomedical Research, Cambridge, MA 02142 Edited by Jasper Rine, University of California, Berkeley, CA, and approved February 4, 2019 (received for review December 7, 2018) The phenotypic consequence of a given mutation can be influ- Recent large-scale comparative screens in many model sys- enced by the genetic background. For example, conditional gene tems, including yeasts, nematodes, Drosophila, mice, and human essentiality occurs when the loss of function of a gene causes cell lines, have revealed an extensive catalog of background ef- lethality in one genetic background but not another. Between two fects, mainly related to differential fitness consequences of well- individual Saccharomyces cerevisiae strains, S288c and Σ1278b, defined loss-of-function mutations across genetically distinct in- ∼1% of yeast genes were previously identified as “conditional dividuals (20–30). In the budding yeast, Saccharomyces cerevisiae, essential.” Here, in addition to confirming that some conditional we carried out a comparative study of systematic gene deletions essential genes are modified by a nonchromosomal element, we in two closely related individuals, S288c and Σ1278b, and showed show that most cases involve a complex set of genomic modifiers. that in this context, ∼1% (57 genes) of all yeast genes are con- From tetrad analysis of S288C/Σ1278b hybrid strains and whole- ditional essential, where the deletion of a given gene is lethal in genome sequencing of viable hybrid spore progeny, we identified one background but not another (23). These data provide an complex sets of multiple genomic regions underlying conditional es- opportunity to systematically dissect the modifiers involved in sentiality. For a smaller subset of genes, including CYS3 and CYS4, background-specific phenotypes related to gene deletion vari- each of which encodes components of the cysteine biosynthesis path- ants. In addition, an important advantage of the yeast model is way, we observed a segregation pattern consistent with a single the availability of a large number of genetically diverse natural modifier associated with conditional essentiality. In natural yeast iso- isolates. So far, the genomes of over 1,000 wild yeast isolates GENETICS lates, we found that the CYS3/CYS4 conditional essentiality can be originating from various ecological and geographical locations caused by variation in two independent modifiers, MET1 and OPT1, have been completely sequenced (31). Combining these re- each with roles associated with cellular cysteine physiology. Interest- sources, the yeast model offers a unique opportunity to explore ingly, the OPT1 allelic variation appears to have arisen independently specific cases of conditional essentiality in two defined genetic from separate lineages, with rare allele frequencies below 0.5%. backgrounds, and then to expand the analysis to the population Thus, while conditional gene essentiality is usually driven by genetic level to discover variant frequency, type, and trait predictability. interactions associated with complex modifier architectures, our anal- Here, we characterized the modifier complexity involved in ysis also highlights the role of functionally related, genetically inde- previously identified conditional essentiality cases between S288c pendent, and rare variants. Significance conditional gene essentiality | background effect | complex modifier interactions | rare variants Genetic background impacts the phenotypic outcome of a mutation in different individuals; however, the underlying enetic backgrounds can impact the phenotypic conse- molecular mechanisms are often unclear. We characterized Gquences of a specific mutation and might constitute an in- genes exhibiting conditional essentiality when mutated in two herent feature of biological traits, complicating our ability to genetically distinct yeast strains. Hybrid crosses and whole- predict individual phenotypes from genomic information (1–9). genome sequencing revealed that conditional essentiality can In many human Mendelian disorders, including cystic fibrosis, be associated with nonchromosomal elements or a single- sickle cell anemia, and neurofibromatosis, even though the modifier locus, but most involve a complex set of modifier causal variant is well established (1, 10–13), individuals carrying loci. Detailed analysis of the cysteine biosynthesis pathway the same causal mutation either do not always develop the dis- showed that independent, rare, single-gene modifiers, related ease, a phenomenon called incomplete penetrance, or do not to both up- and downstream pathway functions, can arise in display the same clinical symptoms, an effect known as variable multiple allelic forms from separate lineages. For several genes, expressivity. Variable penetrance or expressivity of a trait typically we also resolved complex sets of modifying loci underlying conditional essentiality, revealing specific genetic interactions reflects environmental or genetic background influences and sig- that drive an individual strain’s background effect. nificantly impacts the ability to connect genotype to phenotype in natural populations (14). Author contributions: J.H. and C.B. designed research; J.H. performed research; G.T. and While genetic background effects are commonly observed, the G.R.F. contributed new reagents/analytic tools; J.H., G.R.F., B.J.A., and C.B. analyzed data; underlying molecular mechanisms remain mostly unknown. In and J.H., B.J.A., and C.B. wrote the paper. general, genetic variants contributing to background effects are The authors declare no conflict of interest. termed “modifiers.” Identification of critical modifiers is diffi- This article is a PNAS Direct Submission. cult, likely due to the low population frequencies of the modified This open access article is distributed under Creative Commons Attribution-NonCommercial- trait and the heterogenic nature of the modifiers involved (7, 15). NoDerivatives License 4.0 (CC BY-NC-ND). In addition, recent evidence suggests that many genetic back- Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. PRJNA493856). ground effects are caused by highly complex modifier interac- 1To whom correspondence may be addressed. Email: [email protected], brenda. tions, which themselves can be confounded by other genetic and [email protected], or [email protected]. – environmental factors (5, 16 19). As a result, only a handful of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. examples of modifiers involved in human Mendelian diseases, 1073/pnas.1820915116/-/DCSupplemental. notably in cystic fibrosis, have been identified (4, 10, 12, 13). www.pnas.org/cgi/doi/10.1073/pnas.1820915116 PNAS Latest Articles | 1of10 Downloaded by guest on September 26, 2021 and Σ1278b. We mapped the genomic regions involved in a a 2:2 segregation pattern of viable progeny, with the deletion subset of cases and focused on a pair of genes, CYS3 and CYS4, marker cosegregating with the lethal phenotype. Previous work which are involved in the cysteine biosynthesis pathway and are showed that this segregation pattern reflects conditional essen- essential in Σ1278b but not S288c. We characterized and func- tiality associated with cytosolic factors related to the mitochon- tionally validated the modifier underlying the Σ1278b-specific drial genomes and/or the presence of killer viruses (19). For essentiality and expanded our analysis to other natural isolates. 6 other cases, including CYS3 and CYS4, we observed a segre- By surveying a large number of strains, we showed that cysteine gation pattern that was consistent with a single modifier associ- biosynthesis pathway essentiality can be caused by variation in ated with the conditional essential gene. In the single-modifier two independent modifiers, OPT1 and MET1, that are linked to cases, either a 2:2 segregation pattern in the homozygous de- upstream or downstream pathway functions. Sequence analyses letion hybrid or a predominance of tetrads containing four, revealed that allelic variants of the identified modifiers in- three, or two viable spores in the heterozygous deletion hybrid dependently arose from separate lineages and were extremely was observed, indicative of a single-modifier origin (Dataset S1). rare across the population. For the other 20 cases listed in Dataset S1, the segregation patterns in either heterozygous S288c/Σ1278b hybrid alone or Results both heterozygous and homozygous S288c/Σ1278b hybrids to- The Modifier Complexity of S288c/Σ1278b Conditional Gene Essentiality. gether indicate complex modifier origins, which is consistent with We previously compared growth phenotypes of gene deletion mu- the general conclusion of our previous preliminary findings tant collections constructed in two laboratory strains, S288c and basedontetradanalysis(23). Σ1278b, and identified a total of 57 genes as conditional essential, Depending on the interaction patterns among the modifiers, among which 13 were specific to S288c and 44 were specific to the number of modifiers involved in some of the complex cases Σ1278b (23). To select cases for modifier analysis, we first rean- could be inferred
Load more

Recommended publications
  • Molecular Basis for the Distinct Cellular Functions of the Lsm1-7 and Lsm2-8 Complexes
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.22.055376; this version posted April 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes Eric J. Montemayor1,2, Johanna M. Virta1, Samuel M. Hayes1, Yuichiro Nomura1, David A. Brow2, Samuel E. Butcher1 1Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA. 2Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. Correspondence should be addressed to E.J.M. ([email protected]) and S.E.B. ([email protected]). Abstract Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2′,3′ cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines.
    [Show full text]
  • Genetic and Genomic Analysis of Hyperlipidemia, Obesity and Diabetes Using (C57BL/6J × TALLYHO/Jngj) F2 Mice
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Nutrition Publications and Other Works Nutrition 12-19-2010 Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P. Stewart Marshall University Hyoung Y. Kim University of Tennessee - Knoxville, [email protected] Arnold M. Saxton University of Tennessee - Knoxville, [email protected] Jung H. Kim Marshall University Follow this and additional works at: https://trace.tennessee.edu/utk_nutrpubs Part of the Animal Sciences Commons, and the Nutrition Commons Recommended Citation BMC Genomics 2010, 11:713 doi:10.1186/1471-2164-11-713 This Article is brought to you for free and open access by the Nutrition at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Nutrition Publications and Other Works by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. Stewart et al. BMC Genomics 2010, 11:713 http://www.biomedcentral.com/1471-2164/11/713 RESEARCH ARTICLE Open Access Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P Stewart1, Hyoung Yon Kim2, Arnold M Saxton3, Jung Han Kim1* Abstract Background: Type 2 diabetes (T2D) is the most common form of diabetes in humans and is closely associated with dyslipidemia and obesity that magnifies the mortality and morbidity related to T2D. The genetic contribution to human T2D and related metabolic disorders is evident, and mostly follows polygenic inheritance. The TALLYHO/ JngJ (TH) mice are a polygenic model for T2D characterized by obesity, hyperinsulinemia, impaired glucose uptake and tolerance, hyperlipidemia, and hyperglycemia.
    [Show full text]
  • Aneuploidy: Using Genetic Instability to Preserve a Haploid Genome?
    Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Science (Cancer Biology) Aneuploidy: Using genetic instability to preserve a haploid genome? Submitted by: Ramona Ramdath In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Science Examination Committee Signature/Date Major Advisor: David Allison, M.D., Ph.D. Academic James Trempe, Ph.D. Advisory Committee: David Giovanucci, Ph.D. Randall Ruch, Ph.D. Ronald Mellgren, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: April 10, 2009 Aneuploidy: Using genetic instability to preserve a haploid genome? Ramona Ramdath University of Toledo, Health Science Campus 2009 Dedication I dedicate this dissertation to my grandfather who died of lung cancer two years ago, but who always instilled in us the value and importance of education. And to my mom and sister, both of whom have been pillars of support and stimulating conversations. To my sister, Rehanna, especially- I hope this inspires you to achieve all that you want to in life, academically and otherwise. ii Acknowledgements As we go through these academic journeys, there are so many along the way that make an impact not only on our work, but on our lives as well, and I would like to say a heartfelt thank you to all of those people: My Committee members- Dr. James Trempe, Dr. David Giovanucchi, Dr. Ronald Mellgren and Dr. Randall Ruch for their guidance, suggestions, support and confidence in me. My major advisor- Dr. David Allison, for his constructive criticism and positive reinforcement.
    [Show full text]
  • U1 Snrnp Regulates Chromatin Retention of Noncoding Rnas
    bioRxiv preprint doi: https://doi.org/10.1101/310433; this version posted April 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. U1 snRNP regulates chromatin retention of noncoding RNAs Yafei Yin,1,* J. Yuyang Lu,1 Xuechun Zhang,1 Wen Shao,1 Yanhui Xu,1 Pan Li,1,2 Yantao Hong,1 Qiangfeng Cliff Zhang,2 and Xiaohua Shen 1,3,* 1 Tsinghua-Peking Center for Life Sciences, School of Medicine and School of Life Sciences, Tsinghua University, Beijing 100084, China 2 MOE Key Laboratory of Bioinformatics, Beijing Advanced Innovation Center for Structural Biology, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China 3 Lead Contact *Correspondence: [email protected] (Y.Y.), [email protected] (X.S.) Running title: U1 snRNP regulates ncRNA-chromatin retention Keywords: RNA-chromatin localization, U1 snRNA, lncRNA, PROMPTs and eRNA, splicing 1 bioRxiv preprint doi: https://doi.org/10.1101/310433; this version posted April 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Thousands of noncoding transcripts exist in mammalian genomes, and they preferentially localize to chromatin. Here, to identify cis-regulatory elements that control RNA-chromatin association, we developed a high-throughput method named RNA element for subcellular localization by sequencing (REL-seq). Coupling REL-seq with random mutagenesis (mutREL-seq), we discovered a key 7-nt U1 recognition motif in chromatin-enriched RNA elements.
    [Show full text]
  • Structural Basis of Specific DNA Binding by the Transcription Factor
    8388–8398 Nucleic Acids Research, 2019, Vol. 47, No. 16 Published online 21 June 2019 doi: 10.1093/nar/gkz557 Structural basis of specific DNA binding by the transcription factor ZBTB24 Ren Ren1,†, Swanand Hardikar1,†, John R. Horton1, Yue Lu1, Yang Zeng1,2,AnupK.Singh1, Kevin Lin1, Luis Della Coletta1, Jianjun Shen1,2, Celine Shuet Lin Kong3, Hideharu Hashimoto4, Xing Zhang1, Taiping Chen1,2,* and Xiaodong Cheng 1,2,* 1Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, 2 Houston, TX 77030, USA, Program in Genetics and Epigenetics, The University of Texas MD Anderson Cancer Downloaded from https://academic.oup.com/nar/article/47/16/8388/5521786 by guest on 23 September 2021 Center UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA, 3Program in Cancer Biology, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA and 4Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA Received October 13, 2018; Revised June 07, 2019; Editorial Decision June 11, 2019; Accepted June 13, 2019 ABSTRACT INTRODUCTION ZBTB24, encoding a protein of the ZBTB family ZBTB (zinc-finger- and BTB domain-containing) proteins, of transcriptional regulators, is one of four known characterized by the presence of an N-terminal BTB genes––the other three being DNMT3B, CDCA7 and (broad-complex, tram-track, and bric-a-brac)` domain [also HELLS––that are mutated in immunodeficiency, cen- known as POZ (poxvirus and zinc finger) domain] and a tromeric instability and facial anomalies (ICF) syn- C-terminal domain with tandem C2H2 Kr¨uppel-type zinc fingers (ZFs), are a large family of evolutionarily con- drome, a genetic disorder characterized by DNA hy- served transcriptional regulators (1).
    [Show full text]
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts
    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
    [Show full text]
  • The Genetics of Bipolar Disorder
    Molecular Psychiatry (2008) 13, 742–771 & 2008 Nature Publishing Group All rights reserved 1359-4184/08 $30.00 www.nature.com/mp FEATURE REVIEW The genetics of bipolar disorder: genome ‘hot regions,’ genes, new potential candidates and future directions A Serretti and L Mandelli Institute of Psychiatry, University of Bologna, Bologna, Italy Bipolar disorder (BP) is a complex disorder caused by a number of liability genes interacting with the environment. In recent years, a large number of linkage and association studies have been conducted producing an extremely large number of findings often not replicated or partially replicated. Further, results from linkage and association studies are not always easily comparable. Unfortunately, at present a comprehensive coverage of available evidence is still lacking. In the present paper, we summarized results obtained from both linkage and association studies in BP. Further, we indicated new potential interesting genes, located in genome ‘hot regions’ for BP and being expressed in the brain. We reviewed published studies on the subject till December 2007. We precisely localized regions where positive linkage has been found, by the NCBI Map viewer (http://www.ncbi.nlm.nih.gov/mapview/); further, we identified genes located in interesting areas and expressed in the brain, by the Entrez gene, Unigene databases (http://www.ncbi.nlm.nih.gov/entrez/) and Human Protein Reference Database (http://www.hprd.org); these genes could be of interest in future investigations. The review of association studies gave interesting results, as a number of genes seem to be definitively involved in BP, such as SLC6A4, TPH2, DRD4, SLC6A3, DAOA, DTNBP1, NRG1, DISC1 and BDNF.
    [Show full text]
  • Trna Into a Serine-Inserting Trna
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 5680-5684, June 1992 Biochemistry Eight base changes are sufficient to convert a leucine-inserting tRNA into a serine-inserting tRNA JENNIFER NORMANLY*, TRACY OLLICKt, AND JOHN ABELSON Division of Biology, California Institute of Technology, Pasadena, CA 91125 Contributed by John Abelson, February 26, 1992 ABSTRACT Each aminoacyl-tRNA synthetase must func- retrospect, this should have been clear early on when it was tionally dtgh its cognate tRNAs from all others. We have discovered that the amber suppressor allele ofa tRNATrP gene determined the minimum number of changes required to inserts glutamine (22). Recently, we have synthesized amber transform a leucine amber suppressor tRNA to serine identity. suppressor tRNA genes for all 20 isoaccepting groups in E. coli Eight changes are required. These are located in the acceptor (23, 24). Five ofthese tRNAs (Ile-1, Gly-2, Metf, Glu, and Trp) stem and in the D stem. insert glutamine and six others (Ile-2, Arg, Metm, Asp, Thr, and Val) insert lysine (25). This result confirms Kisselev and Correct recognition of tRNAs by aminoacyl-tRNA syn- Frolova's (26) and Schulman and Goddard's (27) long-held thetases (AASs) is a prerequisite for accurate protein synthesis conviction that AASs recognize anticodon nucleotides, and in the cell. Each of the 20 AASs must recognize and ami- there is now much additional evidence to support that notion noacylate its own set of isoaccepting tRNAs and reject all (2). Recently, Schulman and coworkers (28, 29) have inserted others. Elements that facilitate the aminoacylation ofa tRNA different anticodons into the E.
    [Show full text]
  • LSM6 Rabbit Polyclonal Antibody – TA343774 | Origene
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TA343774 LSM6 Rabbit Polyclonal Antibody Product data: Product Type: Primary Antibodies Applications: WB Recommended Dilution: WB Reactivity: Human Host: Rabbit Isotype: IgG Clonality: Polyclonal Immunogen: The immunogen for anti-LSM6 antibody: synthetic peptide directed towards the N terminal of human LSM6. Synthetic peptide located within the following region: MSLRKQTPSDFLKQIIGRPVVVKLNSGVDYRGVLACLDGYMNIALEQTEE Formulation: Liquid. Purified antibody supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose. Note that this product is shipped as lyophilized powder to China customers. Purification: Affinity Purified Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 9 kDa Gene Name: LSM6 homolog, U6 small nuclear RNA and mRNA degradation associated Database Link: NP_009011 Entrez Gene 11157 Human P62312 This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 2 LSM6 Rabbit Polyclonal Antibody – TA343774 Background: Sm-like proteins were identified in a variety of organisms based on sequence homology with the Sm protein family (see SNRPD2; MIM 601061). Sm-like proteins contain the Sm sequence motif, which consists of 2 regions separated by a linker of variable length that folds as a loop. The Sm-like proteins are thought to form a stable heteromer present in tri-snRNP particles, which are important for pre-mRNA splicing.Sm-like proteins were identified in a variety of organisms based on sequence homology with the Sm protein family (see SNRPD2; MIM 601061).
    [Show full text]
  • Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases A, D, and E in Saccharomyces Cerevisiae
    INVESTIGATION Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases a, d, and e in Saccharomyces cerevisiae Marion Dubarry,* Conor Lawless,* A. Peter Banks,† Simon Cockell,‡ and David Lydall*,1 *Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, †High Throughput Screening Facility, Newcastle Biomedicine, and ‡Bioinformatics Support Unit, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom ORCID IDs: 0000-0002-4186-8506 (C.L.); 0000-0003-2478-085X (D.L.) ABSTRACT Three major DNA polymerases replicate the linear eukaryotic chromosomes. DNA polymerase KEYWORDS a-primase (Pol a) and DNA polymerase d (Pol d) replicate the lagging-strand and Pol a and DNA polymerase e DNA replication (Pol e) the leading-strand. To identify factors affecting coordination of DNA replication, we have performed DNA polymerase genome-wide quantitative fitness analyses of budding yeast cells containing defective polymerases. We Profilyzer combined temperature-sensitive mutations affecting the three replicative polymerases, Pol a,Pold,and DIXY Pol e with genome-wide collections of null and reduced function mutations. We identify large numbers of quantitative genetic interactions that inform about the roles that specific genes play to help Pol a,Pold, and Pol e function. fitness analyses Surprisingly, the overlap between the genetic networks affecting the three DNA polymerases does not (QFA) represent the majority of the genetic interactions identified. Instead our data support a model for division of Saccharomyces labor between the different DNA polymerases during DNA replication. For example, our genetic interaction cerevisiae data are consistent with biochemical data showing that Pol e is more important to the Pre-Loading complex than either Pol a or Pol d.
    [Show full text]
  • Identification of Novel Dna Damage Response Genes Using Functional Genomics
    IDENTIFICATION OF NOVEL DNA DAMAGE RESPONSE GENES USING FUNCTIONAL GENOMICS by Michael Chang A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto © Copyright by Michael Chang (2005) Identification of novel DNA damage response genes using functional genomics Doctor of Philosophy, 2005; Michael Chang; Department of Biochemistry, University of Toronto ABSTRACT The genetic information required for life is stored within molecules of DNA. This DNA is under constant attack as a result of normal cellular metabolic processes, as well as exposure to genotoxic agents. DNA damage left unrepaired can result in mutations that alter the genetic information encoded within DNA. Cells have consequently evolved complex pathways to combat damage to their DNA. Defects in the cellular response to DNA damage can result in genomic instability, a hallmark of cancer cells. Identifying all the components required for this response remains an important step in fully elucidating the molecular mechanisms involved. I used functional genomic approaches to identify genes required for the DNA damage response in Saccharomyces cerevisiae. I conducted a screen to identify genes required for resistance to a DNA damaging agent, methyl methanesulfonate, and identified several poorly characterized genes that are necessary for proper S phase progression in the presence of DNA damage. Among the genes identified, ESC4/RTT107 has since been shown to be essential for the resumption of DNA replication after DNA damage. Using genome-wide genetic interaction screens to identify genes that are required for viability in the absence of MUS81 and MMS4, two genes required for resistance to DNA damage, I helped identify ELG1, deletion of which causes DNA replication defects, genomic instability, and an inability to properly recover from DNA damage during S phase.
    [Show full text]
  • Supplementary Table S1. List of Differentially Expressed
    Supplementary table S1. List of differentially expressed transcripts (FDR adjusted p‐value < 0.05 and −1.4 ≤ FC ≥1.4). 1 ID Symbol Entrez Gene Name Adj. p‐Value Log2 FC 214895_s_at ADAM10 ADAM metallopeptidase domain 10 3,11E‐05 −1,400 205997_at ADAM28 ADAM metallopeptidase domain 28 6,57E‐05 −1,400 220606_s_at ADPRM ADP‐ribose/CDP‐alcohol diphosphatase, manganese dependent 6,50E‐06 −1,430 217410_at AGRN agrin 2,34E‐10 1,420 212980_at AHSA2P activator of HSP90 ATPase homolog 2, pseudogene 6,44E‐06 −1,920 219672_at AHSP alpha hemoglobin stabilizing protein 7,27E‐05 2,330 aminoacyl tRNA synthetase complex interacting multifunctional 202541_at AIMP1 4,91E‐06 −1,830 protein 1 210269_s_at AKAP17A A‐kinase anchoring protein 17A 2,64E‐10 −1,560 211560_s_at ALAS2 5ʹ‐aminolevulinate synthase 2 4,28E‐06 3,560 212224_at ALDH1A1 aldehyde dehydrogenase 1 family member A1 8,93E‐04 −1,400 205583_s_at ALG13 ALG13 UDP‐N‐acetylglucosaminyltransferase subunit 9,50E‐07 −1,430 207206_s_at ALOX12 arachidonate 12‐lipoxygenase, 12S type 4,76E‐05 1,630 AMY1C (includes 208498_s_at amylase alpha 1C 3,83E‐05 −1,700 others) 201043_s_at ANP32A acidic nuclear phosphoprotein 32 family member A 5,61E‐09 −1,760 202888_s_at ANPEP alanyl aminopeptidase, membrane 7,40E‐04 −1,600 221013_s_at APOL2 apolipoprotein L2 6,57E‐11 1,600 219094_at ARMC8 armadillo repeat containing 8 3,47E‐08 −1,710 207798_s_at ATXN2L ataxin 2 like 2,16E‐07 −1,410 215990_s_at BCL6 BCL6 transcription repressor 1,74E‐07 −1,700 200776_s_at BZW1 basic leucine zipper and W2 domains 1 1,09E‐06 −1,570 222309_at
    [Show full text]