A study on premature segregation of unreplicated chromosomes 1

A STUDY ON PREMATURE SEGREGATION OF UNREPLICATED CHROMOSOMES KHONG JENN HUI INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2011 A STUDY ON PREMATURE SEGREGATION OF UNREPLICATED CHROMOSOMES KHONG JENN HUI B. Sc. (Hons.), MURDOCH UNIVERSITY A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements I would like to express my earnest thanks, sincere gratitude and appreciation to Professor Uttam Surana for his guidance, insightful and stimulating discussions, as well as valuable advice, which helped sustain my curiosity and passion in this study. My sincere thanks also go out to members of my PhD Supervisory Committee, A/P Yang Xiaohang (IMCB) and Dr. Maki Murata-Hori (TLL), for their constructive comments and encouragement. My deepest gratitu de to Assistant Prof. Lim Hong Hwa, Dr Zhang Tao and Dr. Sihoe SanLing for your continuous help, discussions, guidance and advice, without which this project and thesis would never have become a reality. Special thanks to my lab mates – Dr. Yio WeeKheng, Dr. Idina Shi Yiting, David, HongQing, Joan and all members of CMJ and WY laboratory for sharing, discussions and generous help in various ways. I would like to thank Dr Jayantha Gunraratne and Associate Professor Walter Blackstock (IMCB) for the collaboration on mass spectrometry. I am grateful to Drs. Kim Nasmyth, Frank Uhlmann, Tomo Tanaka, Piatti Simonetta, Matthias Peter for providing me with valuable reagents, yeast strains and constructs which were essential for many experiments. Most importantly, I wish to thank my parents, brother and sister, for their unconditional support, encouragement, prayers and advice. Last but not least, I would like to extend my gratitude to my wife kilyn for your understanding, patience, support and encouragement throughout this study. i Table of Contents Acknowledgements………………………………………………………………… i Table of Contents………………………………………………………………… .ii Summary……………………………………………………………………………vi List of Tables………………………………………………………………………. ix List of Figures……………………………………………………………………… x List f Symbols………………………………………………………………… … .xiii Chapter 1.1 Introduction….………….………………… .1 Introductory Remarks………………………………………. 1.2 1.2.1 Brief overview of cell cycle……………………………… .2 Saccharomyces cerevisiae cell cycle……………………… 1.2.2 Regulation of the transition point between cell cycle phases and cyclin-dependent kinase Cdc28 1.2.3 Regulation of Cdk activity . 1.2.4 Regulation of cell cycle events by checkpoints .7 1.3 Regulation of cell cycle by protein degradation . 1.3.1 The ubiquitin-proteasome system…….………………………9 1.3.2 SCF………………………………………………………… 10 1.3.2.1 SCFCdc4…………… ……………………………………… . 11 1.3.2.2 SCFGrr1……………………………………………………… 13 1.3.2.3 SCFMet30 14 1.3.3 APC…………………………………………………………. 14 1.3.3.1 Substrate specificity of APC……………………………… 15 1.3.3.2 Regulation of anaphase by APC…………………………….17 1.3.3.3 Regulation of mitotic exit by APC………………………… 18 1.3.3.4 Regulation of G1-S by APC……………………….……… 19 1.4 The spindle pole body cycle………… …………………… 21 1.4.1 Spindle Anatomy…………………………………………… 24 1.4.2 Regulation of microtubule dynamics……………………… 27 1.4.3 Regulation of short spindle formation……………………… 32 1.4.4 Regulation of spindle elongation…………………………… 34 1.5 Premature spindle elongation and segregation of unreplicated chromosomes…………………………………………………36 1.6 The G1-M checkpoint pathway………………………………39 1.7 The multiple roles of Cdc6………………………………… 40 ii Chapter Materials and Methods……………….…………………… 50 2.1 Materials…………………………………………………… 50 2.2 Methods…………………………………………………… . 50 2.2.1 Escherichia coli strains and culture conditions…………… . 62 2.2.2 Yeast strains and culture conditions………………………… 62 2.2.3 Cell cycle synchronization………………………………… .63 2.2.4 Yeast transformation…………………………………………64 2.2.5 Isolation of plasmid DNA from yeast……………………… 64 2.2.6 Yeast chromosomal DNA extraction……………………… .65 2.2.7 Southern blot analysis……………………………… .…… .66 2.2.8 Immunofluorescence staining (IF)………………….………. 67 2.2.9 Microscopy………………………………………………… 68 2.2.10 Flow cytometry analysis (FACS)…………………………….69 2.2.11 Preparation of cell extracts for protein analysis……………70 2.2.11.1 Protein extraction using Tri-Chloroacetic Acid (TCA)…… .70 2.2.11.2 Protein extraction using acid-washed glass beads………… .70 2.2.12 Western blot analysis……………………………………… 71 2.2.13 Immunoprecipitation…………………………………………71 2.2.14 PCR-based strategy for fluorescent protein and epitope tagging of yeast genes……………………………………………… .72 2.2.15 Pulse-chase assay…………………………………………….73 2.2.16 Sample preparation for SILAC mass spectrometry………….73 Chapter Premature chromosome segregation in cells with unreplicated chromosomes ………………………………75 3.1 Background………………………………………………… 75 3.2 Results……………………………………………………… 79 3.3 3.2.1 Cells depleted of Cdc6 undergo premature nuclear division in the absence of DNA replication………………………… .79 3.2.2 Premature nuclear division in Cdc6 depleted cells is associated with major mitotic events…………………………………….82 3.2.3 Precocious nuclear division in Cdc6 depleted cells does not require onset of mitosis………………………………………86 3.2.4 Precocious nuclear division in Cdc6 depleted cells can be prevented by dicentric chromosomes……………………… .91 3.2.5 Precocious nuclear division in Cdc6 depleted cells is due to deregulation of spindle dynamics……………………………94 Discussion………………………………………….……… .99 iii Chapter Regulation of spindle elongation by Cdc34……………… 103 4.1 Background……………………………………………… 103 4.2 Results…………………………………………………… . 104 4.2.1 Premature segregation of unreplicated chromosomes in cells lacking Cdc7 and Cdc45…………………………………… 104 4.2.2 Depletion of Cdc6 in cdc34-1 cells fails to promote spindle assembly or spindle elongation………………………….… 109 4.2.3 Ectopic expression of Sic1 and Cdh1 prevent premature spindle elongation in Cdc6 depleted cells……………………… 111 4.2.4 cdc34 and cdc34 cdc6 mutant cells can assemble short bipolar spindles in the absence of Cdh1 or Sic1 but fail to elongate them……………………………………………………… 114 4.2.5 Sic1 degradation promotes Cdh1 inactivation and short spindle assembly……………………………………………… 117 4.2.6 Ectopic expression of microtubules associated proteins induces spindle elongation in Cdc34 deficient cells devoid of Cdh1…………………………………………………… 120 4.2.7 Cdh1 resistant microtubule associated proteins cannot induce complete spindle elongation in cdc34-1 and cdc34-1 cdh1Δ cells………………………………………………………… 124 4.2.8 Loss of Ase1 or Cin8 individually cannot prevent premature spindle elongation in Cdc6 deficient cells…………… . 127 4.2.9 Cdc34 can induce spindle elongation by promoting stability of microtubule associated proteins…………………………… 129 4.2.10 Microtubule associated proteins Ase1 and Cin8 are unstable in cells deficient in Cdc34…………………………………… 137 4.2.11 Cdc34-mediated stabilization of microtubule associated proteins are proteasome dependent………………………………… 140 4.3 Discussion………………………………………… .… Chapter 142 A search for Cdc34-mediated up- or down regulated proteins that promote spindle elongation……… .… 147 5.1 Background……………………………………………… 147 5.2 Results…………………………………………………… 148 5.2.1 Cdc34 promotes up-regulation of the polo-like kinase Cdc5 during premature spindle elongation……………………… 148 5.2.2 Ectopic expression of Cdc5 can induce spindle formation and elongation…………………………………………………. 152 5.2.3 Cdc5 is unstable in the absence of Cdc34 function…… 154 Discussion…………………………………………… . 156 Perspective and future directions…………………… 158 5.3 Chapter iv Bibliography…… .…………………………………………………………… 166 ACKNOWLEDGEMENTS I   TABLE OF CONTENTS II   SUMMARY VI   LIST OF TABLES IX   LIST OF FIGURESX   CHAPTER   INTRODUCTION 14   1.1   Introductory Remarks 14   1.2   Brief overview of cell cycle 15   1.2.1   Saccharomyces cerevisiae cell cycle 15   v Summary High fidelity transmission of the genome to the next generation is imperative for the successful survival of all species. At cellular level, this can be accomplished by accurate duplication and segregation of the genome to two daughter cells during the cell division cycle. In order for chromosome segregation to proceed accurately, the sister chromatids must be attached to the mitotic spindle. Hence, cells have evolved surveillance pathways known as checkpoints to ensure that both spindle cycle and cell cycle progress in a coordinated and timely manner. These checkpoints halt cell cycle progression when damage or defect is detected on chromosomes or spindles and undertake immediate steps to repair detected damages before the cell cycle is allowed to resume. This cell cycle halt can pose extreme danger to cell cycle committed cells (post START) that cannot initiate S phase because spindle forms in the absence of duplicated chromosomes and biorientation will lead to precocious chromosome segregation and genomic instability, a leading cause of aneuploidy. Both DNA Replication and damage checkpoints are known to prevent precocious spindle elongation (i.e. premature chromosome segregation) via regulation of spindle dynamics (Krishnan et al. 2004) (Zhang et al. 2009). A less characterized checkpoint known as the G1-M checkpoint has also been reported to play essential role in prevention of mitosis should cells fail to undergo S phase. Cdc6 has been defined as an important component of the G1-M checkpoint that prevents untimely onset of mitosis when cells fail to initiate DNA replication. This is because yeast cells deficient in Cdc6 fail to initiate DNA replication but proceed to elongate their spindles and segregate the un-replicated chromosomes leading to a “reductional” anaphase (Piatti et al. 1995). Due to the intimate association between chromosome vi segregation and mitosis, it has been proposed that Cdc6 or the G1-M checkpoint prevents onset of mitosis when cells fail to initiate DNA replication (Piatti et al. 1995). Thus far, no other component of this checkpoint pathway has been identified. In Chapter 3, our results suggest that untimely chromosome segregation in the absence of Cdc6 function is not due to premature mitotic entry but is a result of the deregulation of spindle dynamics. Surprisingly, we also find that premature chromosome segregation is a not a property specifically associated with the loss of Cdc6 function but it is a common characteristic of cells (such as cdc7 or cdc45 mutants) that fail to initiate DNA replication. In Chapter 4, our findings implicate Cdc34 (SCF) as a new regulator of spindle dynamics. The clue came to light from the experiment in which spindles were dramatically extended in cdc34-1 cdc6Δ cells when Cdc34 function was restored by a return to the permissive temperature (Figure 27 and 28). This clearly suggests that Cdc34 function is necessary to convert a short spindle to a long spindle and argues that cdc6 mutant cells require Cdc34 function to extend their spindles. In conclusion, the dramatic deregulation of spindle dynamics experienced by cells that are committed to the cell cycle but fail to undergo DNA replication is a result of the interplay of four sequential cellular events: activation of the E3 ubiquitin ligase SCF, destruction of Cdk inhibitor Sic1, inactivation of another ubiquitin ligase APCCdh1and stabilization of microtubule associated proteins. The role of Cdc34 in spindle dynamics is particularly critical during the period between START and S phase in that Cdc34-mediated stabilization of Ase1, Cin8 and Cdc5 (or destabilization of a novel spindle-elongation inhibitor) would cause premature spindle elongation in any cell that traverses START but are unable to initiate S phase. These results suggest vii that initiation of DNA replication saves the cells from potential chromosomesegregation catastrophe. viii phosphorylation by Cdc5 (polo-like kinase) is required to completely inactivate Cdh1. Thus, adequate accumulation of Cin8, Kip1 and Ase1 for bipolar spindle assembly requires sequential phosphorylation of Cdh1 by Cdc28 and Cdc5 (Crasta et al. 2008). 1.4.4 Regulation of spindle elongation As described earlier, microtubules of the mitotic spindle form the structural basis for chromosome segregation. The process of spindle elongation is facilitated by the intrinsic polarity and dynamic properties of microtubules and involves many proteins that modulate microtubule organization and stability. In metaphase, microtubules show high dynamic instability which is essential to aid the “search and capture” of chromosomes for bipolar alignment (Kirschner et al. 1986). Subsequently, microtubules become more stable at the onset of anaphase when motor proteins and other microtubule associated proteins mediate spindle elongation and chromosome separation (Zhai et al. 1995; Mallavarapu et al. 1999; Maddox et al. 2000; KlineSmith et al. 2004). Proteins that contribute to anaphase spindle stabilization and elongation are found to localize to spindle midzone. As the name suggests, the midzone is the middle region of the bipolar spindle where antiparallel interpolar microtubules emanating from the opposite poles overlap. In budding yeast, activation of the phosphatase Cdc14 at the anaphase onset is required for stabilizing microtubule dynamics as persistent high microtubule turnover at anaphase weakens the spindle midzone leading to spindle breakage. Cdc14 activation normally occurs after the dissolution of sister chromatid cohesion (Stegmeier et al. 2002; Sullivan et al. 2003). Cdc14 activation is important for the recruitment of spindle stabilizing proteins such as Stu1 (plus end microtubule associated protein) and Cin8 (plus end BimC family 47 kinesin) to the anaphase spindle for its successful elongation (Higuchi et al. 2005). In addition, localization of midzone organizing protein Ase1 to anaphase spindle is also dependent on its dephosphorylation by Cdc14 (Khmelinskii et al. 2009). Both Cin8 and Ase1 possess microtubule bundling activity that is essential for the maintenance of parallel arrangement of midzone microtubules. Localization of Ase1 also mediate recruitment of other microtubule stabilizing proteins to the midzone, thereby preventing “unzipping” of the spindle and allowing efficient anaphase spindle elongation (Khmelinskii et al. 2009). The finding that anaphase spindles are fragile and fail to elongate above an intermediate length of 4-5µm in ase1Δ cells supports this notion (Fridman et al. 2009). In order to ensure equal partitioning of chromosomes to daughter cells in anaphase, both chromokinesin and cytoplasmic dynein are also indispensable (Hunter et al. 2000). Chromokinesin such as Cin8 is a plus-end directed kinesin localized to chromosome and is essential for chromosome attachment and movement toward the metaphase plate. Cytoplasmic dynein, on the other hand, functions to orient astral microtubules and position the mitotic spindles correctly (Hunter et al. 2000). Thus far we have described the regulations imposed on the mitotic spindle assembly and extension during normal division cycle. However, the process of spindle elongation is largely irreversible. Should these regulations fail under certain cellular circumstances, premature spindle extension can occur causing mis-segregation of chromosomes and genomic instability. In the following section, we examine a few examples of premature spindle elongation, precocious chromosome segregation and conditions that lead to them. 48 1.5 Premature spindle elongation and segregation of unreplicated chromosomes Precise coordination of cell cycle events ensures timely segregation of chromosomes during normal division (Hartwell et al. 1989). For instance, DNA replication must be completed prior to passage through mitosis and all chromosomes should attain biorientation before sister chromatid cohesion can be dissolved or spindle extension can occur. The restrictive conditions that ensure coordination of various cell cycle events are in part imposed by the surveillance mechanism known as the checkpoint controls. Checkpoint controls ensure that a later event is not initiated until prior events are completed successfully. The progression through S phase is monitored by two major checkpoint controls, namely, replication checkpoint- and DNA damage-checkpoints. They prevent premature chromosome segregation in response to incomplete replication and DNA damage respectively (Melo et al. 2002; Osborn et al. 2002). In the budding yeast, Mec1 and Rad53 kinases (orthologues of human ATR and Chk2 kinases) are the main effectors of these pathways (Weinert et al. 1994; Sanchez et al. 1996). Inhibition of DNA replication (by hydroxyurea for instance) in replication checkpoint-defective mutants, such as mec1 and rad53 halt replication fork progression (Enoch et al. 1990; Osborn et al. 2002). However, these cells proceed to elongate their spindles, partition the largely unreplicated chromosomes between the mother and daughter compartments and lose viability rapidly (Krishnan et al. 2004). Since chromosome segregation is conspicuously associated with mitosis, this phenotype is thought to be caused by premature onset of mitosis in the absence of the checkpoint (Enoch et al. 1990). During normal mitosis, chromosome segregation is preceded by the activation of Cdc2/Cdc28 by tyrosine 19 dephosphorylation (tyrosine 15 in fission yeast), activation of APCCdc20, bipolar attachment of kinetochores to 49 spindle microtubules, destruction of securin Pds1, and cleavage of cohesins by the separase Esp1. However, precocious separation of unreplicated chromosomes in HUtreated mec1 or rad53 mutants is not accompanied by many of these major mitotic events (Krishnan et al. 2004). This leads to the proposal that precocious chromosome segregation in replication checkpoint deficient cells is not a consequence of untimely entry into mitosis. Instead, it is a result of premature induction of spindle elongation during early S phase due to accumulation of spindle elongating factors such as Cin8 and Stu2 and downregulation of microtubule destabilizing motor proteins Kip3, thus resulting in the tipping of the balance of forces in favour of elongation (Krishnan et al. 2004; Krishnan et al. 2005). Collectively, the above observations suggest that the replication checkpoint pathway restrains nuclear division in response to inhibition of DNA synthesis by directly modulating spindle dynamics (Krishnan et al. 2004). When treated with HU, Chinese hamster ovary cells also arrest in early S phase. However, upon inactivation of the replication checkpoint by caffeine treatment, they enter mitosis prematurely assemble mitotic spindle and progress through M phase even in the absence of intact, replicated chromosomes (Schlegel et al. 1986; Brinkley et al. 1988; Wise et al. 1997). In S. pombe, mutation in cdc18, cut5 and cdt1 affect initiation of DNA replication and cause a cut phenotype where septation (an event associated with cytokinesis) occurs before nuclear division (Kelly et al. 1993; Saka et al. 1993; Hofmann et al. 1994). The fission yeast gene RUM1 is reported to restrain mitosis in cells arrest prior to START (Moreno et al. 1994). A loss of Rum1 function leads to aberrant mitosis and cytokinesis (the cut phenotype) in the absence of S phase, giving rise to cells with less than 1C DNA (Labib et al. 1995; Jallepalli et al. 1996). Moreover, the orp1Δ replication initiation mutant also arrested 50 in G1 at the restrictive temperature but enters mitosis in the absence of DNA replication (‘cut’ phenotype) (Synnes et al. 2002). The cellular control system that prevents premature onset of mitosis in the event cells fail to initiate DNA replication is termed as G1-M checkpoint. It is distinct from replication checkpoint in that G1-M control is thought to function in cells that are unable to initiate DNA replication at all and is not dependent on Mec1 or Rad53 functions (Toyn et al. 1995). In budding yeast, this less-characterized control system was initially formulated on the phenotypic behavior of cdc6 mutant cells. CDC6 is an essential gene and encodes for an unstable protein required for DNA replication. Cells lacking Cdc6 arrest in late G1 and are unable to initiate DNA replication but undergo precocious chromosome segregation (termed “reductional anaphase”) (Piatti et al. 1995). Since the investigations documented in this thesis pertain to the mechanisms underlying the G1-M checkpoint, we consider this pathway in some detail. 51 1.6 The G1-M checkpoint pathway In budding yeast, the initiation-of-DNA-synthesis mutants (iDS) have been used to investigate the G1-M checkpoint (Toyn et al. 1995). The iDS mutants such as dbf4-1, dbf4-2, cdc7-1, cdc7-43 and mcm3-10 arrest prior to S phase with 1C DNA content and a divided nucleus at restrictive temperature (Toyn et al. 1995). Here the division of the chromatin does not involve separation of sister chromatids but segregation of unreplicated chromosomes (Toyn et al. 1995). In principle, a defect in any of the major and well characterized checkpoints (i.e. replication-, DNA damage- and spindle assembly checkpoint) could account for this phenotype since all of them function to prevent premature initiation progression through mitosis. However, when iDS mutant dbf4 was treated with spindle poison nocodazole and then incubated at restrictive temperature, cells arrested in metaphase and remained viable, indicating that spindle assembly checkpoint is intact in dbf4 mutant cells (Toyn et al. 1995). Similarly, iDS mutants responded normally to HU treatment or DNA damage and arrested with phenotypes similar to that of wild type cells. These observations suggested that precocious segregation of unreplicated chromosomes in iDS cells is not due to the defect in any of the known checkpoints but to a defined novel surveillance system essential for preventing mitosis in cells unable to undergo DNA replication (Toyn et al. 1995). This is also taken to indicate that once cells traverse START, they are committed to mitosis regardless of whether they can duplicate their DNA successfully. G1-M checkpoint is proposed to restrain premature mitosis in this context. 52 1.7 The multiple roles of Cdc6 Eukaryotic replication origins are licensed in late mitosis/early G1 to undergo a single initiation event (Ayad 2005). This coincides with Cdc6 transcription which also occurs primarily in late mitosis and early G1 (Boronat et al. 2007). The origin recognition complex (ORC) first binds to origin DNA, followed by recruitment of Cdc6 and Cdt1 (Liang et al. 1995). Binding of Cdc6 to origins stabilizes interaction of ORC with chromatin. The tight interactions between ORC, Cdc6 and Cdt1 allow recruitment of multiple copies of the minichromosome maintenance Mcm2-7 hexamer (putative replicative helicase) onto each origin (Nishitani et al. 2000; Tanaka et al. 2002). This leads to the formation of pre-replicative complex (pre-RC) and the licensing of the origin for use in the ensuing S phase. Once licensing has occurred and Mcm2-7 are loaded onto DNA, the affinity of Cdc6 for each origin drops and Cdc6 is no longer required for the continued association of Mcm2-7 with DNA (Donovan et al. 1997; Hua et al. 1998; Oehlmann et al. 2004). This encourages the distribution of at least one Mcm2-7 hexamer to each origin and helps to ensure that all origins are licensed. The pre-RC are individually activated and fired at different times in S phase. The firing is executed through phosphorylation of the pre-RC components by Clb5, 6/Cdc28 and the Cdc7/Dbf4 kinase (Elsasser et al. 1999). Once pre-RCs are activated, DNA unwinding occurs and the replication protein A, Cdc45 and DNA polymerase-α are loaded onto the chromatin (Newlon 1997). A second wave of Cdc6 transcription is also observed in late G1 and this ensures the continued presence of Cdc6 during S phase (Lau et al. 2006; Boronat et al. 2007). It was shown in Xenopus egg extracts that the rebinding of Cdc6 to chromatin occurs in S phase after displacing from chromatin when origins were first licensed (Oehlmann et al. 2004). Moreover, Cdc6 can only rebind with high affinity to ORC after progression 53 of replication forks away from the origins (Oehlmann et al. 2004). Using RNA interference, it was shown that when Cdc6 is depleted in synchronous S-phase cells, DNA replication is prolonged leading to mitotic lethality. The Cdc6 depleted S-phase cells also demonstrated fewer newly fired origin (Lau et al. 2006). Despite this, the continued presence of Cdc6 is required for activation of Chk1 that normally occurs as a consequence of replication fork inhibition (Oehlmann et al. 2004). This is reminiscent of results in S. pombe, which showed that Cdc6 homologue Cdc18 is required to activate Cds1 (Chk2) checkpoint kinase in response to the replication inhibitor hydroxyurea (Murakami et al. 2002). These results suggest that Cdc6 has multiple roles in regulating cell cycle progression in addition to its well-documented role in origin licensing. In addition to transcriptional control, the levels of the Cdc6 are controlled by cell cycle-dependent proteolysis (Drury et al. 1997) (Elsasser et al. 1999) (Perkins et al. 2001) (Sanchez et al. 1999) and cellular localization (Piatti et al. 1996) with high accuracy. As described earlier, the production of Cdc6 peaks in late M-early G1 and the protein is very stable during G1 where it accumulates to high levels in nuclei. After the G1-S transition, Cdc6 becomes very unstable and distributed between the nucleus and the cytoplasm more evenly. Cdc6p contains several consensus CDK phosphorylation sites which is important for proteolysis, although this dependence varies with the phase of the cell cycle (Calzada et al. 2000) (Drury et al. 2000) (Elsasser et al. 1999) (Perkins et al. 2001). In yeast, phosphorylation of Cdc6 (or its homologue Cdc18 in S. pombe) by Cdks target it for ubiquitin-dependent proteolysis by SCFCdc4 complex (Baum et al. 1998) (Drury et al. 1997) (Jallepalli et al. 1997) (Kominami et al. 1997). In metazoans, chromatin-bound Cdc6 persists throughout S and G2 phase, whereas it is degraded during G1 by the anaphase promoting complex 54 (Coleman et al. 1996) (Coverley et al. 2000) (Mendez and Stillman 2000) (Petersen et al. 2000). During S and G2 phase, the soluble (non-chromatin bound) Cdc6 is exported out of the nucleus in a Cdk-dependent manner (Saha et al. 1998; Pelizon et al. 2000). Surprisingly, some study showed that non-chromatin-bound Cdc6 may remain in nucleus throughout S phase (Alexandrow et al. 2004). In human cells, phosphorylation of HuCdc6 by Cyclin A/Cdk2 causes export of HuCdc6 from the nucleus after initiation of DNA replication (Petersen et al. 1999). The presence of active Cdc6 degradation mechanisms in S phase versus that in mitosis where the rate of transcription and protein levels of Cdc6 are already very low suggests a need for regulating Cdc6 after the initiation of replication and as a consequence, Cdc6 might play a role later in the cell cycle in addition to its function in the initiation of replication. The notion that Cdc6’s function is necessary in mitosis in addition to being a component of the pre-RC is supported by several pieces of evidence. First, ectopic expression of wild-type Cdc6 to high levels in G2 phase of the cell cycle causes a delay of mitotic entry in both budding and fission yeast (Bueno et al. 1992) (Greenwood et al. 1998) (Perkins et al. 2001). Second, Cdc6 has been reported to inhibit Cdk1/Clb2 directly in promoting timely exit from mitosis (Archambault et al. 2003) (Calzada et al. 2001) (Weinreich et al. 2001). Third, Cdc6 depleted cells undergo a reductional anaphase with unreplicated chromosomes in G1 phase, instead of arresting with short metaphase spindle (Piatti et al. 1995). Fourth, Cdc6 is phosphorylated by Cdk1 on several site. Overexpression of Cdc6 containing Cdk1 phosphorylation site mutant at Threonine 370, Threonine 371 and Serine 372 are lethal in cdc16 background (Elsasser et al. 1999). The fact that cdc16 is one of the components of APC/C (ubiquitination ligase) and it is essential for entry into mitosis, 55 this clearly shows that there is a genetic interaction between replication and mitosis involving Cdc6. Several studies reported inhibition of Cdk1 by high levels of Cdc6 both in vitro and in vivo (Perkins et al. 2001) (Weinreich et al. 2001) (Calzada et al. 2001) (Elsasser et al. 1999) (Basco et al. 1995) (Elsasser et al. 1996) (Calzada et al. 2000) (Mimura et al. 2004). Cdk1 is responsible for activation of APCCdc20 anaphase promoting complex during metaphase by phosphorylating Cdc20. Therefore, it was reasonable to conclude that mitotic delay observed when Cdc6 was overexpressed (described above) was due to lack of Cdk1 activity. However, it was later shown that Cdk1-phosphorylation site mutants of Cdc6 (cdc6 A-C+F and cdc6 A-F) that cannot interact with Cdk1 in vitro and fail to inhibit Cdk1 in vivo were all inhibitors of mitosis (Figure 5) (Boronat et al. 2008). 56 Figure 5: Schematic diagram of Cdc6 protein. Vertical lines depict CDK sites (A-F), and the amino acid numbers at the top of the diagram are given for consensus threonines and serines (Boronat et al. 2008). It was also reported that degradation of mitotic substrates such as Pds1 by APCCdc20 was delayed by expression of the non-Cdk-interacting mutant Cdc6 proteins. Thus, even when there was sufficient Cdk1 to activate APCCdc20, there was another regulation of APCCdc20 by Cdc6 which is independent of Cdk1 (Boronat et al. 2007). Subsequently, genetic analysis showed that the Cdk1/Cdc6 interaction-independent role of Cdc6 in mitosis is dependent on interaction between Cdc6 and Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). The significant observations that deletion of CDC55 abolished the Cdc6-mediated delay in degradation of APCCdc20 substrates, the Cdc6-mediated mitosis and the lethality of Cdc6 overexpression in a cdc16 mutant support the reported interaction between Cdc6 and Cdc55 in mitosis. Several groups also reported premature activation of the mitotic exit network as a result of CDC55 deletion (Minshull et al. 1996) (Wang et al. 1997) (Queralt et al. 2006) (Yellman et al. 2006) (Wang et al. 2006). 57 A proposed model for the roles of Cdc6 in regulating the activity of APCCdc20 in anaphase is depicted in Figure 6. According to the model, newly synthesized mitotic Cdc6 can fully inhibit APCCdc20 function before Cdc20 degradation in late mitosis (Shirayama et al. 1999). Cdc6 may interact with PP2A through the regulatory subunit Cdc55 and recruit PP2A to the APCCdc20 and inhibit it by dephosphorylation, since activation of APCCdc20 requires phosphorylation of both its core subunits (Lahav-Baratz et al. 1995) (Shteinberg et al. 1999) (Rudner et al. 2000) (Rudner et al. 2000) (Kramer et al. 2000) and the APC activator, Cdc20 (Yudkovsky et al. 2000). At this point, the Cdc6 levels may not be sufficient to inhibit Clb/Cdc28 completely as there may be competition between Cdk1 and Cdc55 for binding Cdc6. However, when cells progress to telophase, Cdc6 can inhibit Clb/Cdc28 completely in conjunction with Sic1. This further contributes to the activation of the APCCdh1 and exit from mitosis (Archambault et al. 2003) (Calzada et al. 2001). In addition to this, Cdc6-associated PP2A may dephosphorylate the APCCdc20 contributing to the switch from Cdc20 to Cdh1 since Cdh1 was found to bind unphosphorylated APC core. It is essential to ensure that APCCdc20 is inactivated before Cdc20 degradation by APCCdh1 in order to keep Clb2 levels above a certain threshold. This will prevent premature pre-RC assembly in the presence of increasing levels of Cdc6. Besides this, the stepwise inactivation of APCCdc20 also resulted in reduced Pds1 proteolysis. The undegraded Pds1 population was shown to localize to the mitotic spindle and the spindle pole bodies in mid-anaphase cells and this promote localization of Esp1 to the spindle and coupling exit of mitosis with the earlier completion of anaphase (Jensen et al. 2001). Esp1 has been reported to be involved in mitotic exit by promoting Cdc14 release from the nucleolus (Queralt et al. 2006). In this scenario, it can be envisioned that Cdc6 would be indirectly responsible for maintaining Pds1 level sufficient for 58 promoting Esp1 spindle localization, Esp1-dependent PP2A downregulation and exit from mitosis. According to the model proposed by Boronat and Campbell (2008), Cdc6 plays an essential role in regulating the shift between the two mitotic oscillators, APCCdc20 (negative feedback oscillator) and APCCdh1 (relaxation oscillator) (Cross 2003) (Wasch et al. 2002), thus coordinating the linkage between mitosis and S-phase. 59 Figure 6: A model for the role of Cdc6 in regulating the activity of APCCdc20 in anaphase (Boronat et al. 2008). 60 Rationale for the work presented in our investigation: The assembled mitotic spindle can be lethal to yeast cells if not tightly coordinated with other cell cycle events. Untimely spindle elongation has been reported to cause premature segregation of un-replicated chromosomes in replication-checkpointdefective mutants (such as mec1) treated with DNA replication inhibitors, hydroxyurea (HU). Similarly, cells deficient in Cdc6 also undergo precocious chromosome segregation. Yeast cells deficient in Cdc6 fail to initiate DNA replication but they proceed to elongate their spindles and segregate the un-replicated chromosomes leading to a “reductional” anaphase. Therefore, Cdc6 has been defined as an important component of the G1-M checkpoint that prevents untimely onset of mitosis when cells fail to initiate DNA replication. Phenotypically, replication checkpoint-defective mec1 (+HU) and cdc6 (at restrictive temperature) may appear similar with mono-oriented, randomly segregated unreplicated chromosomes and extended spindle. However, there is an important distinction between the two mutants: while HU-treated mec1 or rad53 cells halt in early S phase with stalled replication forks, cdc6 mutant cells are unable to even initiate DNA replication. In this study, we set out to delineate the mechanism underlying the precocious segregation of unreplicated DNA in cdc6 mutant cells in order to understand the control network that prevent chromosome segregation when cells fail to initiate DNA replication. Our results suggest that untimely chromosome segregation in the absence of Cdc6 function is not due to premature entry into mitosis but is a result of the deregulation of spindle dynamics. This dramatic deregulation is a result of the interplay between four cellular events as cells proceed to DNA replication: activation of the E3 ubiquitin ligase SCF, destruction of Cdk inhibitor Sic1, inactivation of another ubiquitin ligase APCCdh1 (Anaphase Promoting Complex activated by Cdh1) 61 and stabilization of microtubule associated proteins. Surprisingly, we find that premature chromosome segregation is not a property specifically associated with the loss of Cdc6 function but it is a common characteristic of cells (such as cdc7 or cdc45 mutants) that execute the three events but fail to initiate DNA replication. These results suggest that initiation of DNA replication can save the cells from a potential chromosome-segregation catastrophe which the convergence of the three regulatory elements APC, SCF and Sic1 can cause. 62 [...]... progression Therefore, it is mandatory that Cdc20 and Cdh1 activate APC in a sequential manner in mitosis, with APCCdc20 activation at anaphase onset followed by APCCdh1 at the end of mitosis through G1 1. 3.3.2 Regulation of anaphase by APC The anaphase promoting complex (APC) was named for its best-known and most important function in the regulation of anaphase onset Upon completion of S phase, duplicated... substrate ubiquitylation It is also essential that APC can selectively recognize substrates at the correct time for several key events in mitosis- the initiation of anaphase, exit from mitosis and the preparation for the next round of DNA replication The reason why APC-dependent degradation reactions must be tightly regulated is because inappropriate activation of APC can cause fatal coordination-errors... dephosphorylation are essential Phosphorylation of a conserved Threonine -16 9 (similar to Thr -16 7 in S pombe and Thr -16 1 in human) in the T-loop adjacent to the kinase active site is catalyzed by the Cdk-activating kinase (CAK) (Gould et al 19 91) ; (Kaldis et al 19 96) This modification promotes stabilization of Cdc28/Clb complex In addition to this, phosphorylation at the highly conserved Tyrosine -19 (equivalent... APCCdh1 Moreover, Cdh1 phosphorylation also facilitates export of Cdh1 into the cytoplasm contributing to its inactivation (Jaquenoud et al 2002) This explains why Cdc20 is fully synthesized during S and G2 phase when APCCdh1 is inactive (Fang et al 19 98; Prinz et al 19 98; Shirayama et al 19 98; Kramer et al 2000) 1. 3.3.3 Regulation of mitotic exit by APC Upon completion of mitosis, APC also plays an... Fitch et al 19 92; Richardson et al 19 92) 1. 2.3 Regulation of Cdk activity The activity of Cdk1 is regulated at diverse levels As discussed earlier, sequential expression of Cln and Clb cyclins impose control on the Cdk activity at the transcriptional level Cyclin association is insufficient to activate Cdc28 to its full capacity; a number of post-translational events such as phosphorylation and dephosphorylation... manner and, as a result, the activity of mitotic kinase Cdc28/Clb2 is partially reduced (Baumer et al 2000; Yeong et al 2000; 31 Wasch et al 2002) By telophase, it is estimated that 50% of Clb2 has already been degraded by APCCdc20 Surprisingly, a second fraction of Clb2 is resistant to APCCdc20- mediated degradation The partial inactivation of Cdc28 allows the Cdc14 protein phosphatase to dephosphorylate... Cdh1 at the Cdc28 phosphorylation site, leading to activation of Cdh1 (Visintin et al 19 98; Jaspersen et al 19 99) Thus, the second phase of APCCdh1-dependent Clb2 proteolysis begins at the end of mitosis This is followed by the degradation of other APCCdh1 substrates such as microtubuleassociated proteins Cin8, Ase1 and polo kinase Cdc5 (Juang et al 19 97; Charles et al 19 98; Hildebrandt et al 20 01; Castro... to S transition: (1) The APC interacting ubiquitin-conjugating E2 enzyme, UBCH10 is degraded by APCCdh1 This leads to stabilization of APCCdh1 substrates such as cyclin A (Rape et al 2004; Rape et al 2006) (2) Cyclin A activates cyclin-dependent kinase-2 (Cdk2) and phosphorylates Cdh1 to facilitate dissociation of Cdh1 from APC (Lukas et al 19 99; Kramer et al 2000) (3) The phosphorylated Cdh1 is ubiquitylated... Nasmyth 2002) In order to ensure activation of APCCdc20 in mitosis, Cdc20 itself is also tightly regulated The transcription and translation of Cdc20 are completed during S and G2 phase, but Cdc20 can only associate with APC in mitosis upon phosphorylation of APC subunits by mitotic kinases such as Cdk1 and mammalian polo-like kinase -1 (Plk1) (Rudner and Murray 2000) In contrast, phosphorylation of. .. usually act as a signal for proteasome mediated degradation (Nandi et al 2006) Polyubiquitylation at Lys63 may act as a signal for DNA repair but not degradation (Weissman 20 01) Moreover, monoubiquitination of proteins contribute to other pathways such as endocytosis, histone regulation, virus budding and others (Hicke 20 01) In the cell cycle context, SCF (Skp1, Cullin, F box protein complex) and APC . dynamics……………………… 27 1. 4.3 Regulation of short spindle formation……………………… 32 1. 4.4 Regulation of spindle elongation…………………………… 34 1. 5 Premature spindle elongation and segregation of unreplicated chromosomes ………………………………………………36. between START and S phase in that Cdc34-mediated stabilization of Ase1, Cin8 and Cdc5 (or destabilization of a novel spindle-elongation inhibitor) would cause premature spindle elongation in any. biorientation and congress to the metaphase plate. During anaphase, dissolution of sister chromatid cohesion (anaphase A) occurs followed by dramatic elongation of mitotic spindle (anaphase B)