UNIVERSITY OF CINCINNATI Date:__09/27/2004_________________ I, _Katherine L. Lillard___________________________, hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Molecular Genetics, Biochemistry, and Microbiology It is entitled: The BLM helicase functions in alternative lengthening of telomeres. This work and its defense approved by: Chair: Joanna Groden____________ Iain Cartwright__________ Carolyn Price____________ James Stringer___________ Kathleen Dixon___________ THE BLM HELICASE FUNCTIONS IN ALTERNATIVE LENGTHENING OF TELOMERES 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 2004 by Kate Lillard-Wetherell B.S., University of Texas at Austin, 1998 Committee Chair: Joanna Groden, Ph.D. ABSTRACT Somatic cells from persons with the inherited chromosome breakage syndrome Bloom syndrome (BS) feature excessive chromosome breakage, intra-and inter- chromosomal homologous exchanges and telomeric associations. The gene mutated in BS, BLM, encodes a RecQ-like ATP-dependent 3’-to-5’ helicase that presumably functions in some types of DNA transactions. As the absence of BLM is associated with excessive recombination, in vitro experiments have tested the ability of BLM to suppress recombination and/or resolve recombination intermediates. In vitro, BLM promotes branch migration of Holliday junctions, resolves D-loops and unwinds G-quadruplex DNA. A function for BLM in maintaining telomeres is suggested by the latter, since D- loops and perhaps G-quadruplex structures are thought to be present at telomeres. In the present study, the association of BLM with telomeres was investigated. Given the association of BLM with recombination, it was of particular interest to determine the nuclear localization of BLM with respect to telomeres in cells using recombinational pathways for telomere lengthening, termed ALT. Using the telomere repeat protein TRF2 as a telomere marker, we demonstrate that BLM co-localizes with telomeres in cells using ALT, but not in telomerase-positive or primary cells. BLM co- localizes with TRF2 in foci actively synthesizing DNA during late S and G2/M; co- localization is enriched during these phases of the cell cycle when ALT is thought to occur. By immunoprecipitation, BLM associates with telomeres and TRF2 in cells using ALT. In S. cerevisiae, we demonstrate that BLM expression rescues a defect in recombinational telomere lengthening associated with absence of SGS1. These data ii establish a spatial and temporal association of BLM with telomere synthesis in cells using ALT and demonstrate conserved function(s) for BLM and SGS1 in ALT. Additionally, the regulation of BLM activity using telomere substrates was investigated in vitro. We find that TRF1 and TRF2 physically and functionally interact with BLM in vitro. TRF2 stimulates BLM unwinding of telomeric and non-telomeric substrates. Conversely, TRF1 inhibits BLM unwinding of telomeric substrates only. Neither TRF1 nor TRF2 regulate unwinding activity of the UvrD helicase. Finally, BLM helicase activity is stimulated by TRF2 with equimolar concentrations of TRF1, but not when TRF1 is present in molar excess. Based on these data, we present a model for the coordinated regulation of BLM helicase activity by TRF1 and TRF2 at telomeres in cells using ALT. iii Acknowledgments Thank you to members of the Groden lab, past and present. To Joanna for being a wonderful mentor who always had words of encouragement when I needed them most, to Rose for taking care of us all (we would be hopelessly lost without you), to Amod, Chelsea, the Gregs and all the Fries gang for making me relax and have fun (every now and again), to Bill for keeping me company in the late hours my last few months in the lab, to Kathy and Mary for all your support, guidance and wisdom, and a very special thank you to Al… you are an absolute treasure! I’m not sure I would have made it through without your help and companionship. Thank you to my grandmother, my parents, and all my siblings for their continual and everlasting support, to Shonnie for being a friend I can always count on, and last but certainly not least, I thank my husband Seel for being a shoulder to cry on, a friend to laugh with and a partner to rely on for the last ten years and the many years to come. iv Table of Contents Abstract ii Acknowledgements iv Table of Contents 1 Abbreviations 4 List of Tables 6 List of Figures 6 Chapter One. Literature Review Introduction 8 Clinical features of Bloom syndrome. 8 Bloom’s syndrome is characterized by a mutator phenotype. 11 Sensitivity of BS cells to DNA damaging agents. 15 BLM encodes a RecQ-like helicase. 16 Protein partners implicate BLM in DNA repair. 18 BLM is a structure-specific helicase. 19 BLM is regulated by the cell cycle. 22 BLM responds to DNA damage. 23 BLM in DSB repair. 24 Repair of stalled replication forks. 27 BLM is a target and regulator of apoptosis. 27 Other human disorders associated with RecQ deficiency. 29 Sgs1 is a functional homolog of BLM. 31 Murine models for BS. 34 Conclusions 36 Chapter Two. Rationale and Research Objectives 37 1 Chapter Three. Functional association of BLM with recombination-mediated telomere lengthening. I. Introduction 38 II. Materials and Methods Cell lines. 44 Cytogenetics. 44 Cell cycle synchronization, BrdU pulse-labeling, and flow cytometry. 45 Immunostaining. 45 Chromatin immunoprecipitations. 46 In vivo protein co-immunoprecipitations. 46 BLM knock-down. 47 Human telomere length analysis. 47 Yeast expression vectors. 48 Yeast strains and crosses. 48 Yeast telomere length analysis. 49 III. Results Analysis of TAs by FISH analysis is transformed BS fibroblasts. 49 Nuclear localization of endogenous BLM and TRF2. 50 BLM and TRF2 foci are enriched during G2/M and undergo DNA synthesis. 52 Association of BLM with telomeric DNA and TRF2 in vivo. 58 Effects of reducing BLM expression on telomere length in cells using ALT. 60 Rescue of type II telomere lengthening pathway by BLM in sgs1 est2 60 mutant S. cerevisiae. IV. Discussion 64 Chapter Four. Association and regulation of the BLM helicase by TRF1 and TRF2 in vitro. I. Introduction 72 II. Materials and Methods Protein expression and purification. 74 Expression constructs, in vitro transcription and translation (IVTT), 75 and interaction site mapping by in vitro immunoprecipitation. 2 In vitro immunoprecipitation of full length proteins. 77 ELISA assay for detecting TRF1-BLM interaction. 78 DNA substrates. 79 DNase I footprinting. 80 Helicase assays. 80 Gel shift assays. 81 III. Results TRF1 and TRF2 interact with BLM in vitro. 81 BLM unwinds substrates that resemble native telomere conformations. 86 TRF1 and TRF2 oppositely regulate BLM activity on telomeric substrates. 87 TRF2 but not TRF1 affects BLM unwinding of a non-telomeric 3’-overhang. 92 Effects of TRF1 and TRF2 in combination on BLM helicase activity vary 96 with relative concentrations. IV. Discussion 96 Chapter Five. Thesis summary 101 Chapter Six. Bibliography 110 3 Abbreviations aa amino acid ALT alternative lengthening of telomeres APB ALT-associated PML body ATM ataxia telangiectasia mutated protein BIR break-induced replication BLM Bloom’s syndrome protein bp base pair BrdU bromodeoxyuridine BS Bloom’s syndrome BSA bovine serum albumin DSB double-strand break DTT dithiothreitol ECTR extrachromosomal telomeric repeats ELISA enzyme-linked immunosorbent assay FISH fluorescence in situ hybridization GPA glycophorin A HJ Holliday junction HR homologous recombination HPRT hypoxanthine phosphoribosyltransferase HRDC helicase and RNase D C-terminus HU hydroxyurea ICL interstrand cross-links IR ionizing radiation IVTT in vitro transcription and translation LOH loss of heterozygosity MLH1 mutL homologue 1 protein MMC mitomycin C MN micronuclei NBS1 Nijmegen Breakage Syndrome 1 protein 4 ND10 nuclear domain 10 NE nuclear extract NHEJ non-homologous end-joining NLS nuclear localization signal nt nucleotide PD population doubling PML promyelocytic leukemia protein PNB PML nuclear body QR quadriradial RTS Rothmund-Thomson syndrome RMN RAD50-MRE11-NBS1 protein complex RQC RecQ C-terminal domain SCE sister chromatid exchange SDSA synthesis-dependent strand annealing SGS1 slow growth suppressor 1 protein SSA single-strand annealing TA telomeric association TRF terminal repeat fragment TRF1 TTAGGG repeat factor 1 protein TRF2 TTAGGG repeat factor 2 protein UDS unscheduled DNA synthesis UV ultraviolet WRN Werner’s syndrome protein WS Werner’s syndrome 5 LIST OF TABLES Table 1 TAs in metaphase spreads prepared from BS and non-BS lymphoblasts. 14 LIST OF FIGURES Figure 1 Sun sensitivity in BS male. 10 Figure 2 Cytogenetic abnormalities in metaphase spreads from BS cells. 13 Figure 3 BLM protein structure, protein partners and germline BLM mutations. 20 Figure 4 A role for BLM in repair of DSBs and stalled replication fork. 28 Figure 5 BIR-like mechanisms for ALT . 41 Figure 6 Analysis of telomere associations of homologous chromosome arms. 51 Figure 7 BLM co-localizes with TRF2 in cells using ALT. 53 Figure 8 BLM localizes to APBs. 54 Figure 9 Cell cycle regulated association of BLM with PML and TRF2. 56 Figure 10 BLM and TRF2 co-localize with foci of DNA synthesis during 57 late S/ G2/M. Figure 11 BLM co-immunoprecipitates with telomeric DNA and TRF2 from 59 cells using ALT. Figure 12 Knockdown of BLM and TRF analysis in Saos2 cells. 61 Figure 13 Structure of telomeres is S. cerevisiae type I and type II survivors. 63 Figure 14 BLM rescues the type II pathway in sgs1 est2 yeast mutants. 65 Figure 15 A model for the function of BLM in ALT. 71 Figure 16 Recombinant protein purity. 76 Figure 17 BLM directly interacts with TRF1 and TRF2. 84 Figure 18 Mapping of BLM domains that interact with TRF1 and TRF2.