Of complexes and maintenance of genome stability Marek Sebesta, PhD marek.sebesta@ceitec.muni.cz CSB, Ceitec, MU 11-Apr-19 1 Content 1. What is genome stability maintenance? 2. What are the challenges to genome stability? 3. How do cells know genome stability has been compromised? 4. How do cells maintain genome stability? 5. How to study genome stability maintenance? (Case study on Homologous recombination) 2 Genome stability DNA damage response What is genome stability maintenance? DNA repair DNA damage tolerance Chromosome segregation 3 What is genome stability maintenance? It is the ability of living organisms preserve its genetic material in time and across generations. 4 What are the challenges to genome stability? All living mater is constantly exposed to environment that challenges genome stability Exogenous Radiation Diet Stress Endogenous Cellular metabolism DNA replication Transcription Spontaneous modification of the DNA 5 What are the challenges to genome stability? Hoeijmakers, 2001 6 What are the challenges to genome stability? What is more prevalent? Exogenous or endogenous damage? Even-though, historically, exogenous DNA damage was considered to be the prime cause of mutagenesis, recently, as the methodology has progressed, the cellular DNA metabolism pathways (replication and transcription) are being recognised as the more prevalent cause of mutations. 7 What are the challenges to genome stability? Inability to repair properly the damage may lead to cancer, senescence, or apoptosis. 8 What is the difference between a primary lesion and a mutation? 9 What is the difference between a primary lesion and a mutation? primary lesion mutation 10 Transient summary I Terms Genome stability, DNA damage response, DNA repair, DNA damage tolerance denote closely related, yet not interchangeable terms Cells are continuously exposed to wide variety of DNA damage Failure to properly deal with the damage may have fatal consequences to cells 11 How do cells know genome stability has been compromised? Hoeijmakers, 2001 12 How do cells know genome stability has been compromised? The challenges - different types of DNA damage - metabolic state - cell-cycle stage Hoeijmakers, 2001 13 TTA How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA XPC Down-stream events TT RNAPII B CSB Down-stream events TT DNA Pol C helicase RPA Down-stream events14 ATR How do cells react to DNA damage? 15 How do cells react to DNA damage? A simplified picture d’Adda d’Fagagna, 201216 How do cells react to DNA damage? A more comprehensive picture Aguilera and García-Muse, 201317 Transient summary II Cells possess specific factors - sensors - that recognise insults to DNA structure, DNA breaks, or stalled machineries like transcription and replication. The sensors then activate complex signalling pathways that lead to halt of cell-cycle, as well as to decision as of which pathway is to be used; balancing the cell-cycle stage and other needs of the cell. 18 How do cell maintain genome stability? DNA repair is prevalent outside the S-phase, in which DNA damage tolerance is preferred. Hoeijmakers, 2001 19 Curtin et al., 2012 How do cell maintain genome stability? 20 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Sebesta and Krejci, 2016 NHEJ: non-homologous end joining SSA: single strand annealing SDSA: synthesis-dependent strand- annealing DSBR: DSB repair 21 Error-prone Error-free How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Non-homologous end joining NHEJ is an error-prone pathway 22 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Sebesta and Krejci, 2016 23 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination 24 Sebesta and Krejci, 2016 25 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Sebesta and Krejci, 2016 26 Transient summary III Different types of DNA damage are repaired by specific repair pathway The repair is generally error-free, except for NHEJ and SSA In S-phase, cells activate tolerance mechanisms that allow timely completion of DNA replication 27 How to study genome stability maintenance? (Case study on Homologous recombination) www.microbialcell.com Review Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes Hannah L Klein1, *, Kenny K.H. Ang2 , Michelle R. Arkin2 , Emily C. Beckwitt3,4 , Yi-Hsuan Chang5 , Jun Fan6 , Youngho Kwon7,8 , Michael J. Morten1 , Sucheta Mukherjee9 , Oliver J. Pambos6 , Hafez el Sayyed6 , Elizabeth S. Thrall10 , João P. Vieira-da-Rocha9 , Quan Wang11 , Shuang Wang12,13 , Hsin-Yi Yeh5 , Julie S. Biteen14 , Peter Chi5,15 , Wolf-Dietrich Heyer9,16 , Achillefs N. Kapanidis6 , Joseph J. Loparo10 , Terence R. Strick12,13,17 , Patrick Sung7,8 , Bennett Van Houten3,18,19 , Hengyao Niu11, * and Eli Rothenberg1, * 1 New York University School of Medicine, Department of Biochemistry and Molecular Pharmacology, New York, NY 10016, USA. 2 Small Molecule Discovery Center and Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA. 3 Program in Molecular Biophysics and Structural Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA. 4 The University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213, USA. 5 Institute of Biochemical Sciences, National Taiwan University, NO. 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan. 6 Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, OX1 3PU, UK. 7 Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA. 8 Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas 78229, USA. 9 Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616, USA. 10 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA. 11 Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA. 12 Ecole Normale Supérieure, Institut de Biologie de l’Ecole Normale Supérieure (IBENS), CNRS, INSERM, PSL Research University, 28 How to study genome stability maintenance? (Case study on Homologous recombination) Different strategies exist Genetic tools Allow us to identify genes and the relationships among, thereby building a pathway Microscopic tools Give us a glimpse at spacial and temporal relationships of genes of interests Biochemical tools Allow us to understand mechanisms and complex formations within a studied pathway Structural tools Allow us to understand molecular mechanisms at atomic resolution Single molecule techniques Allow us to understand behaviour at of single molecules as compared to bulk biochemical reactions 29 How to study genome stability maintenance? Step1: identify the genes Using a thorough genetic analysis of the isolated mutants, they were able to build a first model of multiple pathways dealing with DNA damage. 30 How to study genome stability maintenance? Step1: identify the genes Using a genetic approach Mimitou and Symington, were able to show for the first time the mechanism by which cells resect the ends of broken DNA. 31 How to study genome stability maintenance? Step2: purify and study the proteins alone Using a purified protein, Patrick Sung was able to show that Rad51 is a bona fide recombinase. 32 How to study genome stability maintenance? Step2: purify and study the proteins in assemblies Using purified proteins, Cejka et al., were able to reconstitute end resection in vitro. 33 How to study genome stability maintenance? Step3: study the proteins in time and space Using life-cell microscopy, Lisby et al., were able to study the spatiotemporal interactions among recombination factors. 34 How to study genome stability maintenance? Step4: study the role of protein complex formation? Using SILAC approaches, Psakhye and Jentsch showed that majority of HR proteins are Sumoylated upon DSBs induction. 35 How to study genome stability maintenance? Step4: study the role of protein complex formation? This Sumo-SIM mediated interactions are trigger timely completion of HR. 36 How to study genome stability maintenance? Step4: study the role of protein complex formation? This Sumo-SIM mediated interactions are trigger timely completion of HR. 37 How to study genome stability maintenance? Step5: study the molecular mechanisms by the means of structural biology Crystal structure of presynaptic filament. 38 How to study genome stability maintenance? Step5: study the molecular mechanisms by the means of structural biology Crystal structure of postsynaptic filament. 39 How to study genome stability maintenance? Step5: study the molecular mechanisms by the means of structural biology By comparing the two structure a detailed, molecular mechanism of the strand exchange reaction can be inferred. 40 How to study genome stability maintenance? Step6: study the molecular mechanisms by the means of single-molecule techniques. 41 How to study genome stability maintenance? Step6: study the molecular mechanisms by the means of single-molecule techniques. 42 Transient summary IV There are different techniques that allow us understand any given pathway The techniques must be combined, in order to get a full picture of the pathway Use whatever technique at hand that will help you answer your scientific question 43 Summary Maintenance of genome stability is a complex endeavour that requires interplay of multiple pathways Cells use sophisticated mechanism in deciding which pathway to use at any given moment Majority of factors responsible for maintaining genome stability acts in complexes, let those be dynamic or not 44