University of Zagreb Faculty of Science Department of Biology Berislav Lisnić RECOMBINOGENICITY AND DISTRIBUTION OF PALINDROMES IN THE YEAST Saccharomyces cerevisiae GENOME DOCTORAL THESIS submitted to the Department of Biology Faculty of Science University of Zagreb for the academic degree of Doctor of Natural Sciences (Biology) Zagreb, 2011. DECLARATION OF AUTORSHIP AND SUPERVISION The research described in this thesis was performed by the author in the Laboratory of Biology and Microbial Genetics at the Faculty of Food Technology and Biotechnology (Zagreb, Croatia), under supervision of dr. sc. Ivan Krešimir Svetec and dr. sc. Zoran Zgaga, as part of the postgraduate program in biology at the Department of Biology, Faculty of Science. ii | Declaration of autorship and superivion ACKNOWLEDGEMENTS Like all scientific endeavours, the work presented here was a collaborative effort that included many people. Preparing, organizing and writing this thesis has been a difficult, long, frustrating - but more than anything else - an exciting and intellectually rewarding adventure. Those who have, directly or indirectly, joined and helped me on this journey have earned my deepest respect and appreciation. First of all, I would like to extend my gratitude to my mentors, Professors Zoran Zgaga and Ivan Krešimir Svetec for their counsel, guidance, patience and encouragement during my undergraduate and graduate years. This thesis would not have seen the light of day without their support. I also record special thanks to my committee members, Professors Višnja Besendorfer and Kristian Vlahoviček for their valuable support and suggestions. Furthermore, I gratefully acknowledge the financial support from the Croatian Ministry of Science, Education and Sports, which made the research described in this thesis possible. It has been a privilege to spend several years at the Laboratory of Biology and Microbial Genetics at the Faculty of Food Technology and Biotechnology, where I have met many people with whom I shared some of the best and some of the worst moments of my PhD adventure. I will always hold our group and lab members, whether former or current, in my memory - particularly Krešimir Gjuračić, Sandra Gregorić, Petar Mitrikeski and Marina Miklenić, who were always ready to lend a helping hand and ear whenever it was needed. The staff of the Ligament polyclinic, Diana, Dora, Hrvoje, Mario, Marinko, Marko, Slavica, Zdravko, and most notably "Il Dottore Medico" - dr. Stjepko Bućan, deserve my sincerest thanks. The knowledge, assistance and friendship Stjepko extended to me over the last couple of years mean to me more than I could ever express. I owe him my heartfelt appreciation. My friends, especially Zvonka Zrnčević, Anita Marjanović, Lucija Barbarić and Igor Obleščuk were never-failing sources of joy, happiness and support when it was most needed. I am very happy to have you around me, and I hope that our friendship will last for many more years to come. Finally, I dedicate this work to the closest members of my family - my mother Jasna, who left us too soon, my wife Vanda and my mother-in-law, Jasna. It is they who, quite often, suffered more from my PhD than myself. Their unconditional love, encouragement, understanding and endless patience were always my driving force and inspiration that allowed me to successfully finish this journey. I owe you everything and I hope that this work will make you proud. Acknowledgements | iii CONTENTS Title page i Declaration of autorship and supervision ii Acknowledgements iii Contents iv Basic documentation card vi Temeljna dokumentacijska kartica vii List of papers viii Abbreviations ix Summary x INTRODUCTION 1 1 | INTRODUCTION TO PALINDROMIC DNA 2 1.1. Nomenclature and definitions 2 1.2. Palindromes are important, but dangerous DNA motifs 4 2 | MECHANISMS OF PALINDROME-DRIVEN GENETIC INSTABILITY 7 2.1. Hairpin-mediated genetic instability 7 2.1.1. Replication slippage 8 2.1.2. Invasion of the 3' end into ectopic homology 11 2.1.3. Nucleolytic processing of the hairpin structure 13 2.1.4. Stalled replication fork reversal 15 2.2. Cruciform-mediated genetic instability 17 2.2.1. Mechanisms of cruciform extrusion 17 2.2.2. Cruciform resolution 20 2.2.3. Center-break palindrome revision 23 3 | MECHANISMS OF PALINDROME FORMATION IN VIVO 25 3.1. Palindromes are formed in vivo as a consequence of DNA metabolism 25 3.1.1. Replication slippage 26 3.1.2. Template switching 27 3.1.3. Fusion of sister chromatids 29 3.1.4. Replication of hairpin-capped DNA molecules 31 4 | PALINDROMES AND HUMAN HEALTH 34 4.1. Palindromes can cause genetic diseases 34 4.1.1. Palindrome-mediated β-thalassemia 34 4.1.2. Palindrome-mediated translocations and associated syndromes 36 4.1.3. Palindromic amplifications and cancer 38 iv | Contents CONTENTS DISCUSSION 41 5 | FACTORS AFFECTING PALINDROME-STIMULATED RECOMBINATION 42 5.1. A 110-bp palindromic insert stimulates recombination regardless of its genomic position 42 5.2. The role of CRP1, MUS81, SAE2 and SPO11 genes in palindrome-induced pop-out recombination 46 5.3. A threshold size for palindrome-induced recombination 47 6 | PALINDROMIC SEQUENCES IN THE YEAST REFERENCE GENOME SEQUENCE 49 6.1. A model for the evolutionary dynamics of palindromic sequences in the yeast genome 51 CONCLUSIONS 53 REFERENCES 54 CURRICULUM VITAE 64 INDIVIDUAL PAPERS 65 Contents | v BASIC DOCUMENTATION CARD University of Zagreb Doctoral Thesis Faculty of Science Department of Biology RECOMBINOGENICITY AND DISTRIBUTION OF PALINDROMES IN THE YEAST Saccharomyces cerevisiae GENOME BERISLAV Lisnić Faculty of Food Technology and Biotechnology Department of biochemical engineering Laboratory of biology and microbial genetics Pierottijeva 6, 10 000 Zagreb, Croatia Genomes of many organisms, from bacteria to mammals, are known to contain significant proportion of palindromic sequences. Although frequently located in cis-acting genetic elements, where they play an important role in the regulation of various cellular processes, palindromes have been recognized as recombinogenic motifs that can provoke various types of genome rearrangements and cause genetic diseases in humans. Employing yeast Saccharomyces cerevisiae as a model organism, this study provides systematic analysis of factors affecting palindrome-stimulated recombination, together with computational tools for distribution analysis of palindromes in DNA sequences. This work establishes for the first time that palindromic sequences become recombinogenic only after they attain a critical length of approximately 70 bp. In addition, the obtained results not only indicate that palindromes conform to specific rules of evolutionary dynamics, but also suggest the existence of a mechanism that suppresses the recombinogenicity of numerous palindromes longer than 70 bp present in the human genome. (64 pages, 18 figures, 1 table, 192 references, original in English) Thesis deposited in National and University Library, Hrvatske bratske zajednice 4, 10 000 Zagreb, Croatia. Keywords: cruciform/genome stability/palindrome/recombination/yeast Supervisor: dr. sc. Ivan Krešimir Svetec Reviewers: dr. sc. Višnja Besendorfer dr. sc. Ivan Krešimir Svetec dr. sc. Kristian Vlahoviček Thesis accepted: July 6th, 2011. vi | Basic documentation card TEMELJNA DOKUMENTACIJSKA KARTICA Sveučilište u Zagrebu Doktorska disertacija Prirodoslovno-matematički fakultet Biološki odsjek REKOMBINOGENOST I DISTRIBUCIJA PALINDROMA U GENOMU KVASCA Saccharomyces cerevisiae BERISLAV Lisnić Prehrambeno-Biotehnološki Fakultet Zavod za biokemijsko inženjerstvo Laboratorij za biologiju i genetiku mikroorganizama Pierottijeva 6, 10 000 Zagreb, Hrvatska Palindromske sekvencije u značajnom su udjelu zastupljene u genomima velikog broja organizama, od bakterija do sisavaca. Iako se često nalaze u raznim cis-djelujućim genetičkim elementima, gdje igraju važnu ulogu u regulaciji raznih staničnih procesa, palindromi su prepoznati kao rekombinogeni motivi koji mogu uzrokovati kako raznovrsne rearanžmane genetičkog materijala, tako i genetičke bolesti kod ljudi. Koristeći kvasac Saccharomyces cerevisiae, provedena je sistematska karakterizacija čimbenika koji utječu na rekombinaciju potaknutu palindromom, te su razvijeni računalni alati za analizu distribucije palindroma u sekvencijama DNA. K tome, u ovom je radu po prvi puta utvrđeno da palindromske sekvencije postaju rekombinogene tek kada dosegnu kritičnu duljinu od približno 70 bp, a ukupni rezultati ne samo da pokazuju kako se palindromske sekvencije pokoravaju specifičnim pravilima evolucijske dinamike, već upućuju i na postojanje mehanizma koji suprimira rekombinogenost brojnih palindroma duljih od 70 bp, koji su prisutni u humanom genomu. (64 stranice, 18 slika, 1 tablica, 192 literaturna navoda, jezik izvornika: engleski) Rad je pohranjen u Nacionalnoj i sveučilišnoj knjižnici, Hrvatske bratske zajednice 4, 10 000 Zagreb, Hrvatska. Ključne riječi: kruciform/stabilnost genoma/palindrom/rekombinacija/kvasac Mentor: dr. sc. Ivan Krešimir Svetec Ocjenjivači: dr. sc. Višnja Besendorfer dr. sc. Ivan Krešimir Svetec dr. sc. Kristian Vlahoviček Rad prihvaćen: 6. srpnja 2011. Temeljna dokumentacijska kartica | vii LIST OF PAPERS This thesis consists of the following papers: o Lisnić B, Svetec IK, Štafa A, Zgaga Z (2009) Size-dependent palindrome-induced intrachromosomal recombination in yeast. DNA Repair 8: 383-389. o Lisnić B, Svetec IK, Šarić H, Nikolić I, Zgaga Z (2005) Palindrome content of the yeast Saccharomyces cerevisiae genome. Current Genetics