UNIVERSITY OF CINCINNATI DATE: 3-22-03 I, Gregory T. Langland , hereby submit this as part of the requirements for the degree of: DOCTORATE OF PHILOSOPHY (Ph.D.) in: The Department of Molecular Genetics, Biochemistry and Microbiology of the College of Medicine It is entitled: INTERACTION BETWEEN THE BLM HELICASE AND THE DNA MISMATCH REPAIR PROTEIN, MLH1 Approved by: Joanna Groden Ph.D. Richard Wenstrup M.D. Jim Stringer Ph.D. Kathleen Dixon Ph.D. Peter Stambrook Ph.D. ii INTERACTION BETWEEN THE BLM HELICASE AND THE DNA MISMATCH REPAIR PROTEIN, MLH1 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, Biochemistry & Microbiology of the College of Medicine 2002 by Gregory T. Langland B.S., University of Cincinnati, 1992 Committee Chair: Joanna Groden, Ph.D. iii `Abstract Bloom’s syndrome (BS) is a rare autosomal recessive disorder that greatly predisposes affected individuals to cancer. Such individuals also are small in size, sensitive to the sun, have immune dysfunction and gross genomic instability. The cytogenetics of BS cells have been extensively studied and have shown increased levels of homologous recombination, quadriradial formations, telomeric associations and chromosome breakage. The gene responsible for BS has been positionally cloned and and encodes a RecQ helicase family member with strand displacement activity that is dependent on ATP and Mg2+. In order to have a greater understanding of BLM helicase function in the cell in regards to DNA replication, recombination and repair, we identified protein- partners of BLM. The C-terminus of BLM identified the DNA mismatch repair protein MLH1 from a yeast two-hybrid screen. In vitro and in vivo immunoprecipitations confirmed the interaction between these two proteins. Using an in vitro mismatch repair assay, BS cell extracts were tested for their ability to correct a single nucleotide mismatch. The BS cell extracts were able to remove the single nucleotide mismatch from the plasmid DNA, demonstrating that the BLM-MLH1 interaction is not necessary to correct a single nucleotide mismatch. To test the hypothesis that MLH1 may regulate the substrate specificity or helicase activity, two different experiments were performed. Gel-shifts were performed plus or minus the presence of MLH1 with different DNA substrates, however MLH1 had no effect on BLM’s ability to bind the different DNA iv substrates. Helicase assays then were performed which demonstrated that MLH1 or the mutL heterodimer modulates the enzymatic activity of BLM by stimulating BLM’s strand displacement activity on the double-overhang (DO) substrate. Finally, we performed experiments with the supF20 mutagenesis system and demonstrated that extracts from BS cells are unable to utilize micro- homology elements within the supF20 gene to restore supF function following the induction of a double strand break (DSB). Additional experiments with the pUC18 mutagenesis system demonstrate that although the efficiency and fidelity of DSB repair by BS extracts are comparable to those of normal extracts when ligatable ends are present, a significant 5-fold increase in mutation rate with BS extracts is observed when terminal phosphates are removed from the DNA substrate that needs repair. Mutant plasmids recovered following DSB repair by BS extracts contain smaller deletions within the lacZα gene not commonly recovered from normal extracts. Colorectal cancer cell lineHCT116 extracts lacking MLH1 were also examined although the efficiency and fidelity of end- joining was similar to control extracts. This suggests that the BLM-MLH1 interaction is not necessary for proper end-joining. In summary this work demonstrates that BS cells lacking the BLM helicase process DSBs differently than normal cells and strongly suggests a role for BLM in aligning micro-homology elements during recombinational events in DSB repair. Disruption of the BLM helicase may lead to replication fork collapses, improper processing of DSBs, genomic instability and ultimately cancer. v vi Acknowledgments Someone once told me anything worth having you will have to work hard and fight for. The completion of my doctoral degree has been no different. It’s more than classwork and benchwork. It has been a learning experience, but let’s just keep it at that. I would like to that everybody that supported me through this effort. Many thanks go to Rick for encouraging me to enter graduate school and gave me the support and encouragement needed to further my career in science. I would like to thank Joanna for having me as a student and all of my committee members for their support and their signature, especially Dr. Peter Stambrook, Dr. Sohaib Khan and my mother for the financial support. Chris H. for showing me that you can give a great thirty-minute seminar with virtually no data. Chris T. for teaching how to make money in graduate school via day-trading and/or fantasy football. Kathy, who will always be my favorite post-doc (Oh, sorry now Assistant Professor). Jenn who Chris T. and I will be fighting for when we have our own labs. Will and Amod will always be remembered as DS-1 and DS-2, they made it fun to be at work. Greg B. for being there and conducting triage after a committee meeting or seminar. I definitely have to thank Al for purifying that nasty protein that I never could. I guess that ten years of protein purification definitely means you have skills. Both Tims were entertaining but I preferred the second one. Chelsea, thanks for the movies and the lunches. James for being there through thick and thin. Lisa, Heather, Mike B.,Dirk, Kevin and Mike for entertaining my wife while I was in graduate school. And last but definitely not least I have to thank Rachel for all the love and support. Now for the questions I hear every day: When are you are graduating ?? Where are you going ?? Is Rachel moving with you ?? To Be Continued…… vii Acknowledgements vii Abstract iv Table of Contents 1 Abbreviations 3 List of Figures 5 List of Tables 7 Chapter 1 – Literature Review 8 Cancer and Genomic Instability 8 Bloom’s syndrome 11 Patient phenotype 15 Positional cloning of BLM 17 Functional motifs of the BLM helicase 19 BLM and PML bodies 23 BLM homologs and orthologs 24 The BLM protein exists as an oligomeric form 26 BLM and WRN knockout mice 27 DNA replication 29 Sensitivity of BS to DNA damaging agents 31 The BASC complex 35 Protein-partners of the BLM helicase 36 Chapter 2 – Thesis Rationale 39 Examining the role of the BLM helicase in mismatch repair and double-strand break repair. Chapter 3 – Material and Methods 40 Cell Culture 40 Reagents and Enzymes 40 Nuclear extract preparation 41 Expression construct generation and characterization 42 Yeast-two hybrid screening 42 Isolation and renaturation of BLM-C 43 IVTT immunoprecipitations 44 Mixed lysate immunoprecipitations 45 In vivo immunoprecipitations 46 Mismatch repair assay 46 supF20 double-strand break repair assay 47 pUC18 double-strand break repair assay 48 Expression and purification of yBLM 49 Preparation of helicase substrates 51 1 Helicase assays 54 Gel-shift assays 55 Chapter 4 – The BLM helicase interacts with MLH1 56 BLM identifies MLH1 in a yeast two-hybrid screen 56 IVTT BLM-C and full-length MLH1 interact 56 Far western assays confirm the interaction between 59 MLH1 and BLM-C BLM and MLH1 interact in vivo 63 DNA mismatch repair activities of BS and control cell 64 extracts are equivalent. Chapter 5– Stimulation of BLM helicase activity by mismatch repair proteins Mismatch repair in E. coli 66 BLM purification and characterization 69 Gel-shift experiments 73 Helicase assays 75 Chapter 6 – The BLM helicase is necessary for normal 82 double-strand break repair Double-strand break repair in mammalian cells 82 In vitro end-joining assay using the supF20 vector 83 In vitro end-joining assay using the pUC18 vector 83 Sequence analysis of pUC18 mutants 90 Examination of the efficiency and fidelity of 94 End-joining from HCT116 cell extracts. Chapter 7- Conclusions and Future Directions 97 Chapter 8- References 113 2 Abbreviations aa- amino acid AT- ataxia telangiectasia BLM- Bloom’s syndrome gene BLM- Bloom’s syndrome protein bp- base pair BS- Bloom’s syndrome DNA-PK- DNA protein kinase DO- double overhang substrate DSB- double-strand break ENU- N-ethyl-nitrosurea HU- hydroxyurea IDL- insertion/deletion loop IP- immunoprecipitation IVTT- in vitro transcription/translation MLH- mutL homologue MMC- mitomycin C NE- nuclear extract NLS- nuclear localization signal nt.- nucleotide PML- promyelocytic leukemia PMS- postmeiotic segregation increased RPA- replication protein A SCE- sister-chromatid exchange SCP- somatic crossover point SDS-PAGE- sodium dodecyl sulfate- polyacrylamide gel electrophoresis UDS- unscheduled DNA synthesis WRN- Werner’s syndrome gene 3 WRN- Werner’s syndrome protein WS- Werner’s syndrome 4 List of Figures Page No. Figure 1. DNA repair genes act as caretakers of the genome. 10 Figure 2. Individual affected by Bloom’s syndrome. 12 Figure 3. BS cells are characterized by high levels of genomic 14 instability. Figure 4. Functional motifs of the BLM helicase. 21 Figure 5. BLM identifies MLH1 in a yeast two-hybrid screen 57 Figure 6. Immunoprecipitations of in vitro transcribed and 58 translated (IVTT) protein products demonstrate the interaction between the C-terminus of BLM and MLH1. Figure 7. Mixed lysate immunoprecipitation demonstrates the 61 interaction between full-length BLM and MLH1 or RPA. Figure 8. Far western assays demonstrate the interaction 62 between the BLM-C terminus and MLH1. Figure 9. BLM and MLH1 interact in vivo. 63 Figure10. DNA mismatch repair activities of BS and 65 HeLa cell extracts are equivalent.