The molecular bases underlying chromosome fragility at Replication Slow Zones in Saccharomyces cerevisiae Nadia Hashash A thesis submitted to University College London in fulfilment of the requirements for the degree of Doctor of Philosophy December 2008 Division of Stem Cell Biology and Developmental Genetics National Institute for Medical Research The Ridgeway Mill Hill London NW7 1AA 1 Declaration “I, Nadia Hashash, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis” Signed Date 2 Abstract Chromosome rearrangements such as translocations and deletions are frequently associated with human cancers. Such rearrangement of the chromosome can be initiated by a DNA break (DSB) that, when inappropriately repaired, may alter chromosome structure. Mammalian common fragile sites are the best-characterised, naturally occurring breakage-prone regions and are deleted or rearranged in many tumour cells. Analogous chromosomal regions also exist in the budding yeast, S. cerevisiae. One example of a yeast fragile site is the replication slow zone (RSZ), so called because the rate of replication fork progression through these regions is slow compared to other regions within the same chromosome. Inactivation of the essential checkpoint kinase, Mec1, in mec1-ts mutants results in replication fork stalling followed by chromosome breakage at RSZs. Interestingly, inhibition of ATR, the mammalian homologue of Mec1, also leads to chromosome instability at common fragile sites, suggesting that the mechanism by which endogenous DSBs are generated is conserved between yeast and mammals. This study aims to enhance our current understanding of common fragile sites using yeast RSZs as a model. First, RSZs were characterised in terms of chromosomal features and determinants in order to identify similarities between RSZs and mammalian common fragile sites and to assess whether yeast RSZs as a suitable system for studying common fragile sites in more complex organisms. Next, the mechanism underlying chromosome fragility at RSZs was investigated by examining the contribution of various chromosomal processes to break formation at these sites. These include: (i) replication fork restart processes (ii) spindle force, (iii) chromosome condensation and decatenation, (iv) chromosome segregation, and (v) cytokinesis. The analyses suggest that chromosome breakage within RSZs requires the actions of the evolutionarily conserved type II topoisomerase and condensin complex. 3 Finally, factors involved in maintaining the stability of RSZs were also explored. The Rrm3 helicase and Psy2 phosphatase complex were found to suppress chromosome breakage at RSZs in a manner dependent on Tel1, another checkpoint kinase. These findings suggest that Tel1 is somehow implicated in chromosome stability at RSZs. The findings presented in this study further our understanding of RSZs and the molecular bases governing their fragility, providing some insight into the mechanism of fragile site instability in mammals. 4 Table of Contents Declaration .................................................................................................. 2 Abstract ........................................................................................................ 3 Table of Contents ........................................................................................ 5 List of Figures .............................................................................................. 11 List of Tables ............................................................................................... 14 List of Abbreviations .................................................................................. 15 Acknowledgements ..................................................................................... 19 Chapter 1: Introduction ..................................................................... 20 1.1 General introduction .......................................................................... 20 1.2 Fragile sites ......................................................................................... 21 1.2.1 Rare Fragile sites .................................................................................... 21 1.2.2 Common fragile sites .............................................................................. 22 1.2.3 Fragile sites in budding yeast ................................................................. 25 1.3 Overview of the eukaryotic cell cycle ............................................... 28 1.4 Chromosome duplication .................................................................. 29 1.4.1 Origins of DNA replication .................................................................... 29 1.4.2 Initiation of DNA replication ................................................................. 30 1.4.3 Progression of the replication fork ......................................................... 35 1.4.4 Cohesion ................................................................................................. 37 1.4.5 Replication termination .......................................................................... 38 1.5 Mitosis ................................................................................................. 39 1.5.1 Chromosome condensation .................................................................... 40 1.5.2 Anaphase onset ....................................................................................... 42 1.5.3 Exit from Mitosis .................................................................................... 45 1.6 Cytokinesis .......................................................................................... 48 1.7 Checkpoints that regulate fragile site stability ................................ 49 1.8 The DNA damage checkpoint ........................................................... 50 1.8.1 Activating the DNA checkpoint ............................................................. 51 1.8.2 Amplifying the signal ............................................................................. 54 1.8.3 Effectors of DNA checkpoints ............................................................... 54 5 1.8.4 DNA repair mechanisms ........................................................................ 56 1.8.5 Turning off the checkpoint ..................................................................... 60 1.9 Responding to replication stress ....................................................... 61 1.9.1 The S-phase checkpoint .......................................................................... 61 1.9.2 Inhibition of late origin firing ................................................................. 63 1.9.3 Stabilisation of stalled forks ................................................................... 65 1.9.4 Restarting stalled forks ........................................................................... 66 1.9.5 Restarting stalled forks as a model for fragile site expression ............... 68 1.10 DNA damage and/or replication checkpoint response proteins acting at common fragile sites ................................................................... 71 1.11 The spindle assembly checkpoint ................................................... 72 1.11.1 What is sensed? .................................................................................... 72 1.11.2 Checkpoint proteins .............................................................................. 72 1.11.3 Effectors of the SAC ............................................................................ 75 1.12 Additional roles of checkpoint proteins ......................................... 75 1.13 Aims of this project .......................................................................... 77 Chapter 2: Materials and Methods ................................................... 78 2.1 Commonly used buffers and solutions ............................................. 78 2.2 Bacterial techniques ........................................................................... 78 2.2.1 Bacterial strains ...................................................................................... 78 2.2.2 E. coli media and growth conditions ...................................................... 79 2.2.3 E. coli transformation ............................................................................. 79 2.2.4 Purification of E. coli plasmid DNA ...................................................... 80 2.3 Yeast techniques ................................................................................. 80 2.3.1 Yeast media and growth conditions ....................................................... 80 2.3.2 Mating yeast strains ................................................................................ 81 2.3.3 Tetrad dissection ..................................................................................... 83 2.3.4 Determination of cell density ................................................................. 83 2.3.5 Growth synchronisation ......................................................................... 83 2.3.6 Fluorescence activated cell sorting (FACS) ........................................... 84 2.3.7 Temperature sensitivity assays ............................................................... 84 2.3.8 Yeast transformation .............................................................................. 84 6 2.3.9