Open Research Online The Open University’s repository of research publications and other research outputs A yeast model of Bloom’s syndrome. Thesis How to cite: Chakraverty, Ronjon (1999). A yeast model of Bloom’s syndrome. PhD thesis The Open University. For guidance on citations see FAQs. c 1998 Ronjon Chakraverty https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.00010208 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk A Yeast Model of Bloom’s Syndrome Thesis submitted for the degree of Doctor of Philosophy Dr/Rbnjon Chakraverty Bsc. MB ChB MRCP (UK) Dip.RCPath. Institute of Molecular Medicine, Oxford. Sponsoring Establishment for The Open University October 23rd 1998 ProQuest Number: U531845 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U531845 Published by ProQuest LLO (2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT Bloom's syndrome (BS) is a rare inherited disorder associated with growth retardation, immunodeficiency and a predisposition to cancers of all types. At a cellular level BS is associated with marked chromosomal instability, as manifest by interchanges between homologous chromosomes and sister chromatids. BS arises as a consequence of defects in a DNA helicase, BLM, which is a member of the RecQ family of helicases. Defects in other RecQ family helicases, derived from prokaryotic and lower eukaryotic cells, also result in a loss of genomic integrity. This suggests that this group of helicase subfamily share a degree of functional conservation. In this thesis, the validity of using the budding yeast, S.cerevisiae, as a model system for BS has been assessed. In this organism, the structural homologue of the BLM protein is Sgslp. Deletion of Sgslp is associated with mitotic hyperrecombination and defective chromosome segregation. Deletion of SGSl also suppresses the slow growth and hyperrecombination phenotype of top3 mutants, which are defective in topoisomerase III. Ectopic expression of a wild type SGSl gene in sgslAtopSA mutants reinduced the slow growth defect, and this effect was reproduced when the human BLM gene was expressed. This result suggested firstly, that the yeast and human proteins share functional similarities and secondly, that the interaction between the RecQ family helicases and topoisomerase m is highly conserved. On this basis, the remainder of this thesis focussed upon the role of topoisomerase HI. Deletion of the S.cerevisiae TOPS gene leads to a slow growth phenotype accompanied by an accumulation of cells with a late S/G2 content of DNA. topSA mutants exhibit a RAD24/RAD9-doponà&al delay in the G2 phase, suggesting a role for topoisomerase m in the resolution of abnormal DNA structures/damage arising during S-phase. Consistent with this notion, topSA strains are defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA damaging agents and are sensitive to killing by a variety of DNA damaging agents, including ultra-violet light, y- rays and the alkylating agent MMS. This S-phase checkpoint defect was not associated with a failure to induce expression of DNA damage response genes. Consistent with an S-phase specific role for topoisomerase HI, expression of the TOPS mRNA is activated in the late G1 phase, and DNA damage checkpoints operating outside of S-phase are unaffected by deletion of TOPS. All of the phenotypic consequences of loss of topoisomerase m function were suppressed by deletion of SGSl. These data implicate topoisomerase m and, by inference, Sgslp in a checkpoint role in response to DNA damage arising during S-phase. A model to explain the role of these proteins during DNA rephcation is proposed. CONTENTS Title page 1 Abstract ii Table of Contents iii Acknowledgements vi Abbreviations vii Chapter 1 Introduction 1 1.1 Introduction 2 1.2 The'recombinational repair'disorders 2 1.3 Human disorders associated with defects in RecQ family helicases 13 1.4 The RecQ subfamily of DNA helicases 22 1.5 Functional relationships between topoisomerases and RecQ family 35 helicases 1.6 Conclusions 50 Chapter 2 Materials and Methods 51 Chapter 3 Assessment of the ability of BLM to complement 78 S.cerevisiae sgslA m utants 3.1 Introduction 79 3.2 Results gl 3.3 Discussion 98 Chapter 4 An evaluation of the cell cycle defect in 103 S.cerevisiae top3A m utants 4.1 Introduction 1Q4 4.2 Results IO9 m 4.3 Discussion 131 Chapter 5 The role of S.cerevisiae topoisomerase III in mediating checkpoint responses to DNA damage and replication blockade 5.1 Introduction 136 5.2 Results 139 5.3 Discussion 172 Chapter 6 Conclusions 177 B ibliography 186 IV To my parents ACKNOWLEDGEMENTS The last three years have felt very much like a non-ending obstacle course: one obstacle is negotiated only for another to block the way. It has without doubt been the most challenging and difficult period of my medical career. I hope that this time spent in laboratory research has taught me two things: first, the ability to critically appraise medical or scientific research and second, a real appreciation of just how hard it is to be successful in pursuing a scientific career. I would like to thank all those people who have helped me during this time. I would like to thank both my supervisors, Ian Hickson and Adrian Harris, for their effective and consistent guidance during this period of study. Ian has always been available to help solve problems and to offer practical solutions. He has devoted countless hours to my cause and I hope that it wasn’t completely fruitless. I would also like to thank ChrisNorbury for his technical advice and his offer to read manuscripts or listen to oral presentations. I would also like to acknowledge Noel Lowndes for lending me laboratory space, and Maria-Angeles de la Torres for teaching me how to do Northern's and for providing me with unpublished reagents. For technical support I would like to thank Sally Davies, Jonathan Kearsey, Phillip North, Charles Redwood and Paul Watt, aU of whom offered ideas on how to overcome various technical problems. I espescially would like to thank Carina Vessey and Richard Isaacs who, throughout the last three years have been such good friends. Most of all I must thank Sally, my best friend, who has had to bring up our two little boys, Oliver and Joe, almost single-handed. She has been so supportive and loving, and I shall never forget this. Finally, I would like to thank the Medical Research Council and the Imperial Cancer Research Fund for their financial support. VI ABBREVIATIONS ATP adenosine triphosphate ATM ataxia telangiectasia mutated BLM Bloom’s syndrome mutated bp base pairs cdc cell division cycle cDNA complementary DNA CHEF clamped homogeneous electric fields DNA deoxyribonucleic acid DSB double strand break dsDNA double-stranded DNA E.coli Escherichia coli EDTA ethylenediaminetetraacetic acid FACs fluorescence-activated cell sorting HEPÉS 4-(2-hydroxyethyl)-l-piperazine ethane sulfonic acid HU hydroxyurea kb kilobase pairs kDa kilodaltons mRNA messenger RNA MMS methyl methane sulfonate œ optical density PCR polymerase chain reaction RNA ribonucleic acid RNase ribonuclease rpm revolutions per minute SCE sister chromatid exchange SDS-PAGE SDS-polyacrylamide gel electrophoresis S. cerevisiae Saccharomyces cerevisiae S. pombe Schizosaccharomyces pombe SDS sodium dodecyl sulphate vu ssDNA single-stranded DNA TBE tris/borate/EDTA buffer Tris tris (bydroxymethyl)-aniinomethane ts temperature sensitive v/v volume for volume w/v weight for volume WRN Werner’s syndrome mutated vm Chapter 1 Introduction 1.1 Introduction The loss of genomic integrity is an invariant property of tumour cells. The study of inherited cancer-prone disorders has shed considerable light upon the means by which human cells maintain genomic integrity. Interpretation of the accumulating data in this field has been facilitated by comparative analyses in prokaryotic and eukaryotic model systems. These studies have demonstrated, for the most part, a remarkable degree of functional conservation of the individual players and pathways involved in the maintenance of genome stability in all organisms. In the last decade, the cloning of genes defective in the disorders ataxia telangiectasia, xeroderma pigmentosum and hereditary non-polyposis colon cancer has generated important information regarding the role of cell cycle checkpoints, nucleotide excision repair and mismatch repair, respectively, in preventing genome instability and suppressing cancer (Shiloh, 1997; Wood, 1997; Jiricny, 1996). The prior dissection of many aspects of these processes in model systems was such that, at least in the cases cited above, putative functions could be assigned to many of the cloned human genes almost as soon as their protein coding sequences were available. In this thesis, a yeast model has been employed in order to understand in greater detail, the basis for the loss of genomic integrity associated with the cancer-prone disorder. Bloom's syndrome (BS). This disorder arises as a consequence of a defect in a DNA helicase, which has structural similarity to a family of helicases found in prokaryotes and lower eukaryotes, the so-called RecQ family (Ellis et al., 1995).