UNIVERSITY OF CINCINNATI _____________ , 20 _____ I,______________________________________________, hereby submit this as part of the requirements for the degree of: ________________________________________________ in: ________________________________________________ It is entitled: ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ Approved by: ________________________ ________________________ ________________________ ________________________ ________________________ BLM is a Suppressor of DNA Recombination A dissertation submitted to the Division of Research and Advanced Studies Of the University of Cincinnati In partial fulfillment of the Requirements for the degree of Doctorate of Philosophy (Ph.D.) In the Department of Molecular Genetics, Microbiology, and Biochemistry Of the College of Arts and Sciences 2002 by Joel E. Straughen B.S., The Ohio State University, 1985 M.D., University of Cincinnati, 2002 Committee Chair: Joanna Groden, Ph.D. i ABSTRACT Bloom’s syndrome (BS) is a rare, recessive chromosome breakage disorder characterized by small stature, sun sensitivity, facial erythema, immunodeficiency, female subfertility, male infertility, and a predisposition to a variety of cancers. When this body of work was started, the gene for Bloom’s syndrome (BLM)hadyettobe identified. This work presents characterization of the genomic region at BLM and the identification of BLM. With the cloning of the gene, the answers to a number of questions could be investigated. Additional chapters present data that demonstrate that increased genomic instability and recombination are the result of loss of function of the Bloom’s syndrome gene product. Somatic cells from BS individuals are characterized by a high frequency of chromatid exchanges both between and within chromosomes, as well as by a high mutation rate at specific loci. DNAs from BS and normal clonal cell lines were first examined for alterations at microsatellite repeat loci. Alterations in size microsatellite repeats were observed at a 10-fold increase in frequency in BS clones compared to normal clones. A contiguous representation of 2-Mb region that contains the BLM gene was generated. YAC and P1 clones from the region were identified and ordered using genetic markers in the region along with newly developed sequence tagged sites from radiation- reduced hybrids, polymorphic dinucleotide repeat loci, and end-sequences of YACs and P1s. The physical map, and DNA markers derived from it, was instrumental in ii identifying BLM. With the gene identified, the genomic structure was determined and a rapid DNA screening test was developed for the identification of BlmAsh,themost common mutation in BS. To determine whether BLM can suppress recombination we over-expressed BLM in separate cell lines capable of identifying recombination or frameshift events. However, no significant difference was noted between cells transfected with BLM and those transfected with vector alone. Finally, we established a mouse model of BS using homologous recombination to disrupt mouse Blm. Genotyping offspring from heterozygous parents did not identify any offspring homozygous for the knockout allele, suggesting embryonic lethal phenotype. In long-term studies, heterozygosity for Blm increases tumor formation compared to wild- type littermates. iii Acknowledgements This adventure started in 1992 when I left my previous career as an electrical engineer where I was modifying and testing airborne radar jamming equipment at various military installations around the country. I left this job, intending at the time only to go to medical school. However while waiting for acceptance into medical school, I was fortunate to get a position as a research assistant with a new faculty member at the University of Cincinnati. Dr. Joanna Groden showed me techniques to manipulate DNA, to alter the course of a cell. She peaked my interest in molecular biology and provided an opportunity for me to contribute to our knowledge of cancer. I appreciate her tutelage, helping me to ask the right questions and giving me the tools to research the answers. I am grateful for being able to work in Dr. Groden’s laboratory. Post-doctoral fellow Therese Tuohy and later, Kathy Heppner Goss were always willing and able to answer my endless questions concerning laboratory technique and experimental design. I appreciate all the social events we enjoyed as a lab. I will not soon forget the competitiveness of Chris Tzrespacz nor the comic relief so often provided by Chris Heinen, both former students in the Groden lab. Thanks to all the past and present members of the Groden, I enjoyed it, thoroughly. During my first year in medical school, I found myself spending more time in the lab than studying. Not that I was disappointed with medical school, but rather excited by molecular genetics and the powerful laboratory techniques to investigate diseases. I was hooked, and after my first year of medical school, I was fortunate to be accepted into the Physician Scientist Training Program (PSTP) at the University of Cincinnati. This small group of extremely motivated people has been a tremendous treasure and resource. My iv thanks go to all members of the program, faculty, staff, and students. Thanks for letting me part of something great. Leadership has always been notable in this program. At the time of my matriculation from this program, Dr. Leslie Myatt had the helm. We all appreciated his common sense approach to solving our problems. Special mention goes to Dr. Judith Harmony, who began the PSTP at the University of Cincinnati and encouraged me to apply. Judy is the most enthusiastic supporter that anyone can have. She always wore the coolest, wildest earrings. Thank you Judy! Thanks to my thesis committee, Drs. Groden, Menon, Stringer, Stambrook, and Fagin. Their insight and experience in science and in life was always useful and often entertaining. Finally, I give my thanks and love to all of my family. To my dad, Dr. William J. Straughen, who always knew I could do this, and my mom, who really wanted me to be sure that I wanted to do this. Thanks. This quest would have been unlikely to occur or at least far less interesting without my wife, Nancy and my two girls, Tegan and Sloane. Thanks for patients and support. I owe you one. I will always owe you one. Joel E. Straughen v TABLE OF CONTENTS Abstract. i Acknowledgements. iv Table of Contents. 1 List of Figures. 2 List of Tables. 5 List of Abbreviations. 6 Chapter 1. Literature Review. 16 Chapter 2. Thesis Rationale. 46 Chapter 3. Materials and Methods. 48 Chapter 4. Microsatellite Instability in Bloom’s Syndrome. 74 Chapter 5. Physical Mapping of the Bloom’s Syndrome Locus. 91 Chapter 6. Cloning and Identification of BLM. 108 Chapter 7. The Genomic Structure of BLM. 133 Chapter 8. A Rapid Method for Detecting the Predominant Ashkenazi 140 Jewish Mutation in the Bloom’s Syndrome Gene. Chapter 9. Frameshifting and Recombination in Cell Lines 147 Over-Expressing BLM. Chapter 10. Creating a Mouse Model of Bloom’s Syndrome. 156 Chapter 11. Discussion. 178 Literature Cited. 192 1 List of Figures Number Title Page 1 SCE in Bloom’s Syndrome Cells 14 2 Generation of Secondary Cell Lines 74 3 Dinucleotide Repeat Instability in BS Cells 77 4 Trinucleotide Repeat Instability in BS Cells 79 5 Smaller and Larger Novel Alleles in a Single 81 Clone at a One Microsatellite Locus 6 Mismatch Repair in BS Cells 83 7 2-Mb Physical Map of the Bloom’s Syndrome 96 Locus 8 Cross-reference Map of the BS locus 98 9 Fluorescent In Situ Hybridizatin 100 10 Long-range Restriction Map 103 11 Step Taken to Construct the Physical Map 107 12 Radiographic Evidence Supporting a 250-kb Region 109 for the BLM Locus 13 Somatic Cross Point Mapping 112 14 Helicase Motifs in BLM 114 15 RecQ homologues 116 16 Human Homologues to BLM 117 17 Northern Blot Analysis of BLM 119 2 18 SSCP Analysis of Persons with BS 122 19 Mutations Identified in Persons with BS 124 20 Dendorgram of RecQ Family Members 129 21 Genomic Structure of BLM 137 22 Intron/Exon Boundaries of BLM 138 23 Schema for Screening for BLMAsh 144 24 Demonstration of Detecting BLMAsh 145 25 G11 Cell Line Can Measure Frameshift Events 148 26 Photograph of the Colorimetric Assay in G11 Cells 150 27 Results of Overexpressing BLM in G11 Cells 151 28 FSH Cells Can Measure Recombination 152 29 Results of Overexpressing BLM in FSH Cells 154 30 BLM Knock-out Mouse Construct 158 31 PCR Screening of Embryonic Stem Cells 160 32 Gross Examination of Mice Blm-/- fetuses 163 33 Histology of Lymphoma from a Blm+/- mouse 165 34 Micronuclei Formation in Blm+/- Mice Compared 167 to Blm+/+ Mice 35 Mating Strategy for Blm+/- and ApcMin/+ Mice 168 36 Comparison of Tumor Number in Blm+/- and Blm+/+ Mice 169 37 Histology of Intestinal Tumors in Blm+/-, ApcMin/+ Mice 171 38 Strategy and Results of Determination of Loss of 172 Heterozygosity in Tumors from Blm+/-, ApcMin/+ Mice 3 39 Model for the Role of Blm in tumor progression 177 in ApcMin/+ Mice 40 Model for Function of BLM in Suppressing SCE 187 41 Model for Function of BLM in Suppressing DSBs 189 4 List of Tables Table Title Page 1 Oligonucleotides Used to Amplify Microsatellite Repeat Sequences 76 2 STSs Used in the Physical Mapping of BLM 94 3 Polymorphic Microsatellites Used or Isolated in the Mapping of BLM 101 4 Restriction Fragments in Kilobases Identified by Hybridization of 105 Probes