Developmental Regulation of DNA Replication in Drosophila melanogaster By
Eugenia Agnes Park B.S., Biochemistry Washington State University
Pullman, WA, 1998
Submitted to the Department of Biology
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy in Biology
At the A MASSACHUSET-S INwTyIE
Massachusetts Institute of Technology OF TECHNOLOGY
Cambridge, MA
SEP 13 2006
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gust2006
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© 2006 Eugenia Agnes Park. All rights reserved.
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The author hereby grants to MIT permission to reproduce and to distribute Publicly paper and electronic copies of this thesis document in whole or in part.
Signature of A uthor... ... ... . ... ... ...
Department of Biology August 17, 2006
C ertified by. .. ..•..y ... ...
Terry L. Orr-Weaver Professor of Biology Thesis Supervisor A ccepted by ... ... .... ... ... ... ... 0 T Stephen Bell
Chair, Committee on Graduate Students Department of Biology
Developmental Regulation of DNA Replication in Drosophila melanogaster
By
Eugenia Agnes Park
Submitted to the Department of Biology
in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology
ABSTRACT
In mitotic cell cycles, the genome must be replicated fully in each cell cycle to ensure the normal complement of chromosomes. Failure to replicate chromosomes fully or a failure to limit
replication to once-per-cell-cycle may lead to aneuploidy and genomic instability. Variants of the archetypal mitotic cell cycle, utilizing conserved cell cycle machinery, are employed during metazoan development to achieve different aims. Endocycles, in which the cell cycle proceeds without complete mitosis, generate polyploidy and are commonly employed to increase metabolic capacity and cell size. D. melanogaster follicle cell gene amplification, in which bi-directional replication occurs in the absence of detectable gap phases, serves to produce large amounts of eggshell proteins and may also serve to regulate transcription. During D.
melanogaster embryogenesis, mitotic cell cycles, endocycles and cell cycle exit occur concurrently. We undertook a screen to identify factors affecting developmentally regulated, variant cell cycles during D. melanogaster embryogenesis. We identified a class of mutants with apparently polyploid cells in normally diploid tissues indicating a failure to maintain mitotic cycles. In this class of mutants, we identified and characterized new mutants in pavarotti and tumbleweed, pav3CS3 and tumrn32a-2
.These mutants displayed phenotypic defects consistent with
failures in cytokinesis. In particular, tum32
a-2o displayed multinucleate cells and abnormal
telophase spindles. We also describe the identification, cloning and characterization of the first cyclinE mutant to undergo aberrant gene amplification, cyclinE1 36. We observed a novel gene
amplification defect, dramatically increased replication fork progression in cyclinE'f36/cyclinEPs
and cyclinE136/cyclinEP28 follicle cells implicating CyclinE in the regulation of replication fork speed.
Thesis Supervisor: Terry L. Orr-Weaver Title: Professor of Biology
ACKNOWLEDGEMENTS
I would like to thank my advisor Terry Orr-Weaver for her guidance and support. Her love for science and her integrity are inspiring. Thank you. Thanks also to the Orr-Weaver Lab, quite frankly, THE GREATEST LAB ON THE PLANET and special thanks to Tamar Resnick with whom I've had many helpful discussions. I would also like to thank my thesis committee for guidance. Thanks to David MacAlpine, Cary Lai and Steve Bell for consistently helpful and informative discussions and to my friends Eunice and Piyush who always had something
interesting to say about science. Last but not least, special thanks to my little sister June who has been unflaggingly supportive.
TABLE OF CONTENTS
Chapter One:
Introduction:
The Developmental Regulation of DNA replication... ...
- Regulation of the mitotic cell cycle...8...
- Regulation of the endocycle...9
- G ene amplification... ... 12
- Cyclin/Cdk and regulation of the Pre-Replication Complex... ... 15
R eplication Initiation... ... 16
- Eukaryotic origins of replication...16
- Epigenetic determinants of metazoan origin activity...18
- The Pre-Replication Complex...19
- A molecular mechanism for Mcm2-7 loading at the Pre-Replication Complex...20
- Mechanisms of Cyclin/Cdk regulation of the Pre-Replication Complex...20
- The transition to replication...24
R eplication Elongation... ... 25
- Replication Fork Progression...26
Sum m ary... ... 27
R eferences ... ... ... 29
Chapter Two:
New mutants affecting developmentally regulated cell cycles during Drosophila embryogenesis Sum m ary... ... 37Introduction ... 38
R esu lts ... 46
- A screen for mutations affecting developmental cell cycle regulation...46
- Class V mutants have large nuclei in the nervous system and epidermis...52
- Characterization and mapping of Ir8 and 3C157...55
- Characterization of 3C53 and 32a-20...58
- 3C53 and 32a-20 are alleles ofpavarotti and tumbleweed/RacGAP50...61
D iscussion ... . . ... 75
- The identification of mutants that affect developmental regulation of the cell cycle...75
- New mutants that affect mitotic cell cycles in the nervous system...76
- tum3•2a-2 disrupts cytokinesis and shows central spindle defects...77
M aterials and M ethods... 79 R eferences...8 1
Chapter Three:
The characterization of new cyclinE mutants that increase replication fork progression during gene amplification
Sum m ary... ... 85
Introduction... ... ... 86
R esu lts...9 1 - A new mutation in cyclinE that displays previously undescribed defects in gene am plifi cation... ... 91
- cyclinEJl36/cyclinEPz8 and cyclinEIP6/cyclinEPz8 have expanded amplified regions at DAFC-66D, DAFC-30B and DAFC-34B...94
- Increased replication fork progression is not due to prior cell cycle defects... 103
- Increased replication fork progression in cyclinEf36/cyclinEPz 8 follicle cells may reflect increased replication fork speed...110
- Polyteny is intact in cyclinE"36/cyclinEPz follicle cells...115
- cyclinE'f36 displays a dominant defect in replication fork progression... .... 118
- Double Parked and the MCM complex localize as double bars in cyclinEf36/cyclinEPz8 follicle cells... ... 122
D iscussion... ... 126
M aterials and M ethods... ... 134
References... ... 137
Chapter Four:
Conclusions and perspectives Conclusions and perspectives... ... 143CHAPTER ONE
The Developmental Regulation of DNA Replication
DNA replication is developmentally regulated during Drosophila melanogaster
development (Claycomb et al., 2002; Smith and Orr-Weaver, 1991). Endocycles, in which alternating S and G phases produce polyploidy, and replication-based gene amplification, are employed to increase tissue size and metabolic output. Drosophila endocycles occur during embryogenesis, larval development and oogenesis and are regulated by conserved replication factors. Gene amplification, in which successive replication initiation events and elongation occur in the absence of detectable gap phases, occurs during oogenesis. Drosophila follicle cell gene amplification is employed to produce high copy numbers of chorion, or eggshell, genes (Spradling, 1981). Like endocycles, follicle cells employ conserved cell cycle regulators (Claycomb and Orr-Weaver, 2005).
The following chapter reviews developmental regulation of endocycles and gene amplification. In addition, current knowledge on mechanisms controlling DNA
replication initiation and elongation are reviewed.
Regulation of the mitotic cell cycle
The archetypal cell cycle consists of G 1, S, G2 and M phases and is regulated by Cyclin/Cdk complexes consisting of a regulatory Cyclin subunit and a Cdk. Different Cyclin/Cdk complexes act in different phases of the cell cycle. In S. cerevisiae, Cyclin subunits confer phase-specificity on a single Cdk, Cdc28. The G1 Cyclins, the Clns, are required for passage through START which signifies commitment to the cell cycle and S-phase entry. The G2 Cyclins, the Clbs, act in S, G2 and M S-phases. Degradation of the
Clbs is required for exit from mitosis (Reed, 1992). Unlike yeast, metazoans possess multiple Cdks (Sherr and Roberts, 1999; Solomon, 1993). CyclinE/Cdk2 activity is required for the G1/S transition. In mammals, CyclinA/Cdk2 regulates S-phase progression. CyclinA/Cdkl and CyclinB/Cdkl activities are required for mitosis.
CyclinD/Cdk4 and CyclinD/Cdk6 mediate the convergence of growth factor signaling on the cell cycle and are thought to indirectly regulate S-phase entry by potentiating
CyclinE/Cdk2 activity (Perez-Roger et al., 1999; Sherr and Roberts, 1999). Cyclin/Cdk is regulated redundantly during the cell cycle. Mechanisms for regulating Cyclin/Cdk activity include oscillatory Cyclin expression, regulatory phosphorylation, inhibition by Cyclin/Cdk inhibitors (CKI) and the targeted degradation of Cyclins.
Regulation of the endocycle
Endocycles consist, minimally, of discrete S and G1 phases with one round of DNA replication occurring per endocycle (Smith and Orr-Weaver, 1991). Some
endocycling tissues show vestiges of mitosis ranging from chromosome condensation to nuclear divisions (Edgar and Orr-Weaver, 2001) and utilize mitotic machinery to regulate Cyclin/Cdk activity. During mitotic cycles, the APC, an E3 ubiquitin ligase, marks Cyclins for degradation by the 26S proteasome. Mutations in morula, an APC subunit, lead to ectopic spindle formation and chromosome condensation in Drosophila nurse cells undergoing endocycles, suggesting that mitotic regulators are expressed at low levels in these cells (Kashevsky et al., 2002; Reed and Orr-Weaver, 1997). APC activity may be required for establishing Drosophila embryonic endocycles by clearing mitotic Cyclins. Mutants in an APC coactivator, fizzy-related (fzr), fail to initiate embryonic
endocycles and fzr is developmentally regulated by Notch in a mitotic to endocycle switch during oogenesis (Schaeffer et al., 2004; Sigrist and Lehner, 1997). In addition, mitotic cyclin transcription is shut off during Drosophila embryonic endocycles (Weiss et al., 1998). Together, these observations suggest that endocycles arise from
downregulation of mitosis specific regulators and support the idea that endocycles are a modification of the archetypal mitotic cell cycle.
Some endocycles show no vestiges of mitosis and may achieve oscillatory
Cyclin/Cdk activity through means that don't involve the APC or mitotic Cyclins. Cyclin E/Cdk2 is important for mammalian endocycles. Double knockouts of the two murine isoforms of CyclinE, CCNE1 and CCNE2, die midgestation due to defects in the placenta without any apparent defects in embryos. Specifically, the trophoblast giant cells do not
attain normal levels of polyploidy. This may reflect the particular importance of CyclinE/Cdk2 in regulating mammalian endocycles (Berthet et al., 2003; Geng et al., 2003; Parisi et al., 2003). In Drosophila, CyclinE is required for embryonic endocycles
(Knoblich et al., 1994).
cyclinE transcription corresponds with S-phase in the Drosophila endocycling tissues and is key to developmental regulation of endocycles (Knoblich et al., 1994; Lilly and Spradling, 1996). cyclinE is not expressed in intervening gap phases, suggesting that endocycles are driven by pulses of cyclinE (Knoblich et al., 1994; Lilly and Spradling,
1996). Low CyclinE levels between pulses of cyclinE transcription are required for replication. Ectopic expression of cyclinE in Drosophila larval endocycling tissues inhibits S-phase (Follette et al., 1998; Weiss et al., 1998).
Cyclical cyclinE transcription is important for endocycles and a biphasic
oscillator, consisting of the E2F1/Rbf transcription factor and the CyclinE/Cdk2 inhibitor Dacapo, has been postulated to regulate cyclinE transcription (Edgar and Orr-Weaver, 2001). In the endocycling tissues of the Drosophila embryo, the E2F1 transcription factor regulates cyclinE expression as part of a G1/S transcriptional program (Asano and
Wharton, 1999; Duronio and O'Farrell, 1994; Royzman et al., 1997). A positive feedback loop, in which CyclinE/Cdk2 hyperphosphorylates and inactivates Rbf, the E2F1
repressor, poses a mechanism for upregulating cyclinE. CyclinE activates expression of the CyclinE/Cdk2 inhibitor dacapo, which encodes a CIP/KIP family member, thereby inhibiting CyclinE/Cdk2 (de Nooij et al., 2000; Lane et al., 1996). Dacapo presents a mechanism for downregulating CyclinE protein levels by inhibiting CyclinE/Cdk2 activity allowing accumulation of hypophosphorylated Rbf, which shuts off E2F1-mediated cyclinE expression. Together, these regulatory loops may define a biphasic oscillator for endocycles that ensures alternating S and G1 phases in endocycles (Edgar and Orr-Weaver, 2001).
Both developmental signaling and growth signaling play key roles in endocycle progression. During Drosophila embryogenesis, endocycles occur in a precise pattern corresponding to developmental stage (Smith and Orr-Weaver, 1991). Notch signaling is involved in the mitotic to endocycle switch in Drosophila follicle cell endocycles and induces transcriptional changes in dacapo,fzr and string (Deng et al., 2001; Schaeffer et al., 2004; Shcherbata et al., 2004). Drosophila larval endocycles are inhibited by nutrient deprivation (Britton and Edgar, 1998) and Ras and c-myc overexpression in mitotically proliferating cells induce growth and hasten the G1/S transition, suggesting that growth
signaling also regulates the cell cycle (Johnston et al., 1999; Prober and Edgar, 2000). A study of CIn3 translation in S. cerevisiae suggests another mechanism for coupling growth to the endocycle. Cln3 mRNA carries a 5' untranslated open reading frame (ORF) that reduces the efficiency of Cln3 translation, suggesting that Cln3 protein synthesis reflects cellular ribosomal content (Polymenis and Schmidt, 1997). cyclinE mRNA carries a number of 5' untranslated ORFs and mutations in eIF4A, a translation initiation factor, confer defects in DNA replication (Galloni and Edgar, 1999). These results
suggest that cyclinE may act as a growth sensor in metazoan cells.
Gene amplification
Gene amplification is a replication-based method for increasing the output of a gene. Replication based gene amplification occurs in Amphibians, Insects and the marine ciliate Tetrahymena thermophila. Amphibians, Tetrahymena and Pterygotan insects amplify rDNA through extrachromasomal mechanisms. Dipteran insects employ gene amplification to increase the copy number of structural genes required for egg maturation (Claycomb and Orr-Weaver, 2005).
The best-characterized example of gene amplification occurs in follicle cells during Drosophila oogenesis. During Drosophila follicle cell gene amplification, repeated replication initiation events, occurring without detectable gap phases, generate high copy numbers of chorion genes required for eggshell formation (Spradling, 1981). These replication initiation events generate multiple tandem replication forks that move
bi-directionally and generate an onion-skin structure (Fig. 1). Gene amplification occurs at a handful of genomic sites known collectively as the Drosophila Amplicons of follicle
Figure 1. Gene amplification in Drosophila follicle cells occurs by repeated replication initiation events that generate tandem replication forks moving away from a central region.
Gene amplification is visualized by BrdU-incorporation at follicle cell amplicons. (A-C) BrdU incorporation in single CantonS wildtype follicle cells in stage 10B (A), stage 11 (B) and stage
13 (C) egg chambers. In stage 10B egg chambers, follicle cells show 4-6 BrdU-labeled foci
corresponding to replication at follicle cell amplicons. In the example shown (A), 4 BrdU-labeled foci are evident and the major focus (arrow) corresponds to repeated replication initiation events generating bi-directional, tandem replication forks at DAFC-66D. Replication initiation and the generation of tandem replication forks in stage 10B are diagrammed to the right of (A) with BrdU-labeling shown in pink. In stage 11 (B), only DAFC-66D (arrow) continues to gene amplify. The last replication initiation events at DAFC-66D occur during this stage although replication elongation continues through stages 12 and 13. By stage 13, BrdU-labeling reveals a double bar structure at DAFC-66D (arrow). The double bar corresponds to BrdU incorporated by opposing sets of tandem replication forks -diagrammed to the right of (C) with BrdU
incorporation in pink. Tandem replication forks have moved apart following cessation of replication initiation events in stage 11 prior to the start of BrdU-labeling and incorporate BrdU at distant sites.
stage 10B stage 11 stage 13
0--~·lc~\· ~11"1~· ~I~ ~c~s CeLIIIII~ IIIIILe ~cllrrr,
Cells, or DAFC. During gene amplification replication initiation events are
developmentally regulated: DAFC-66D undergoes replication initiation events in stages 10B and 11 and only replication elongation occurs from stage 12 on (Fig. 1). (Claycomb et al., 2004; Claycomb et al., 2002). Replication elongation can be visualized
cytologically by BrdU-incorporation at elongating replication forks (Fig. 1) (Claycomb et al., 2002).
Gene amplification is regulated by CyclinE. High CyclinE levels are thought to restrict genomic replication (Calvi et al., 1998). DAFC escape this control and conserved replication factors, components of the Pre-Replication Complex (Pre-RC) localize to the amplicons (Asano and Wharton, 1999; Austin et al., 1999; Claycomb et al., 2002; Royzman et al., 1999; Whittaker et al., 2000).
Cyclin/Cdk and regulation of the Pre-Replication Complex
Low Cyclin/Cdk activity in G of mitotic and endocycles is required for assembly of the Pre-Replication Complex (Pre-RC) at origins of replication. The Pre-RC is an assembly of the Origin Recognition Complex (ORC), Double Parked/Cdtl, Cdc6 and the Mcm2-7 complex (Dutta and Bell, 1997). Regulated assembly of the Pre-RC in G
licenses origins of replication for firing in S-phase. Once-per-cell-cycle assembly
enforces once-per-cell-cycle genomic replication. Cyclin/Cdk phosphorylation of the Pre-RC regulates nuclear compartmentalization, chromatin association, protein stability, and activity of complex members (Findeisen et al., 1999; Hendrickson et al., 1996; Ishimi et al., 2000; Labib et al., 1999). Redundant, Cyclin/Cdk dependent mechanisms for
multiple Pre-RC components must be disrupted to achieve significant re-replication (Gopalakrishnan et al., 2001; Nguyen et al., 2001). More recently, the direct binding of Cyclins to replication factors (Clb2 to Cdc6 and Clb5 to Orc6) in S. cerevisiae have presented a novel Cyclin-dependent mechanism for inhibiting Pre-RC assembly (Mimura et al., 2004; Wilmes et al., 2004).
Gene amplification is different from the mitotic and endocycle in that there are no detectable gap phases. CyclinE levels are constitutively high throughout the nucleus (Calvi et al., 1998). In spite of this, the Pre-RC localizes to DAFC. Orc2, Orcl, Orc5, DUP/Cdtl and Mcm2-7 localize to amplifying foci (Asano and Wharton, 1999; Austin et al., 1999; Claycomb et al., 2002; Royzman et al., 1999; Whittaker et al., 2000) and mutations in Pre-RC components Orc2, DUP/Cdtl, and Mcm6 result in reduced gene amplification and corresponding thin eggshells (Landis et al., 1997; Schwed et al., 2002; Whittaker et al., 2000). CyclinE/Cdk2 may be locally regulated or a molecular switch may function to allow reiterative replication initiation. In addition, an ortholog of the S-phase kinase Dbf4/Cdc7, which is required for replication initiation in mitotic cells, may be required for gene amplification. Mutants in chiffon, which shows homology to the Cdc7 kinase cofactor Dbf4, displays reduced gene amplification (Landis and Tower, 1999).
Replication Initiation
Eukaryotic origins of replication
Genetic screens for autonomously replicating sequences (ARS) led to the
et al., 1979; Struhl et al., 1979). Origins of replication bind ORC, which nucleates Pre-RC assembly thereby defining sites of replication initiation. S. cerevisiae origins of replication are atypical for eukaryotes in that they consist of several well-defined sequence elements (10-20 bp) spread over an approximate 200 bp interval. The A-element consists of the 11 bp ARS consensus sequence (ACS) and is necessary but not sufficient for origin activity. B-elements contribute to origin activity to varying degrees but as a group, are essential (Dutta and Bell, 1997). Like S. cerevisiae, S. pombe and Yarrowia lipolytica origins demonstrate ARS activity but unlike S. cerevisiae, they lack consensus sequences (Vernis et al., 1997; Vernis et al., 1999). These origins are AT rich and approximately 1 kb in size and S. pombe Orc4p carries a specialized binding domain that recognizes AT rich sequences (Chuang and Kelly, 1999).
Metazoan replicators are more complicated. Generally, yeast replicators
encompass a single, preferred replication initiation site or origin of replication. Metazoan origins may encompass several replication initiation sites without a predominant
preferred origin of replication and range in size from 1 to 50 kb (Bielinsky and Gerbi, 2001). Comparisons of these replicators have not yielded consensus sequences or conserved sequence features. Metazoa show developmental plasticity and cell-type specificity in origin usage and this may indicate the epigenetic nature of metazoan origins (Blow, 2001). In Drosophila gene amplification at DAFC-66D, the ORC binding sites are sequence defined at ACE3 (Amplification Control Element 3) and orif3 (Austin et al., 1999).
Epigenetic determinants of metazoan origin activity
Chromatin structure, including covalent modification of DNA and modifications of chromatin packaging proteins, regulate origin usage. CpG methylation inhibits
replication initiation: Methylated DNA does not bind ORC in Xenopus egg extracts, CpG islands are correlated with metazoan origins, and methylase deficient cell lines show less localized replication (Delgado et al., 1998; Gilbert, 2004; Harvey and Newport, 2003a; Rein et al., 1999). In addition, chromatin structural changes such as histone acetylation affect replication initiation. In Drosophila, during follicle cell gene amplification, mutations in the histone deacetyltransferase Rpd-3 result in nuclear localization of ORC and genomic replication, suggesting that histone acetylation promotes origin usage. Consistent with this, tethering Rpd-3 or polycomb to gene amplifying origins of replication reduces replication while tethering the acetyltransferase Chameau increases replication (Aggarwal and Calvi, 2004).
Transcription may promote an open chromatin configuration that is favorable for replication initiation. Numerous transcription factors affect Drosophila follicle cell gene amplification. Mutants in E2F1, E2F2, DP, Rbfl, Myb, Mipl20 and Mipl30 lead to gene amplification defects (Beall et al., 2002; Bosco et al., 2001; Cayirlioglu et al., 2003; Royzman et al., 1999). Mutants in E2F2, Myb and Mipl30 disrupt origin specification in Drosophila follicle cells. These mutants undergo genomic replication rather than gene amplification (Beall et al., 2004; Beall et al., 2002; Cayirlioglu et al., 2001; Cayirlioglu et al., 2003). Transcription factors may impinge on gene amplification by recruiting
chromatin re-modeling factors, which influence origin usage (Aggarwal and Calvi, 2004; Bosco et al., 2001).
Topological factors may also affect replication initiation. Eukaryotic replication origins are distributed in intergenic regions (Wyrick et al., 2001). Active transcription may generate negative supercoils in intergenic regions, which may play a role in origin specification. Interestingly, Drosophila ORC has been shown to bind preferentially to negatively supercoiled DNA (Remus et al., 2004).
The Pre-Replication Complex
The order of Pre-Replication Complex assembly was determined by immunodepletion experiments in Xenopus and through experiments utilizing temperature-sensitive alleles in yeast. In summary, ORC is required for Cdc6 and DUP/Cdtl binding to origins, which are required, in turn, for loading of the Mcm2-7 complex, the putative replicative helicase (Maiorano et al., 2000; Tanaka et al., 1997). Pre-RC assembly is regulated by Cyclins by Cyclin/Cdk phosphorylation and inhibitory binding by Cyclins (Mimura et al., 2004; Wilmes et al., 2004). The Pre-RC is conserved in yeast, flies, mammals, Xenopus and plants, suggesting that mechanisms for replication are conserved across eukaryotes. In addition, Pre-RC components are necessary for mitotic cell cycles, endocycles and Drosophila follicle cell gene amplification indicating conserved mechanisms across different modes of DNA replication.
A molecular mechanism for Mcm2-7 loading at the Pre-Replication Complex
In S. cerevisiae, ORC has been shown to bind DNA cooperatively with Cdc6 in an ATP-dependent manner (Speck et al., 2005). In Xenopus, Cdc6 is known to stabilize ORC binding to chromatin (Harvey and Newport, 2003b). In Xenopus, DUP/Cdtl
localizes to origins after Cdc6 (Tsuyama et al., 2005). Cdc6 ATPase activity is ORC- and origin DNA-dependent and functions at a step prior to ORC ATP hydrolysis, which is required for Mcm2-7 loading (Bowers et al., 2004). Loss of Cdc6 ATPase activity stabilizes DUP/Cdtl at origins and prevents Mcm2-7 loading. As is the case in S. cerevisiae, ORC and Cdc6 ATPase activities are required for Mcm2-7 loading (Harvey and Newport, 2003b). These observations suggest a molecular machine that loads Mcm2-7 onto replication origins in an orderly manner in the following scheme: ORC and Cdc6 bind cooperatively to origins. DUP/Cdtl localizes after Cdc6 binding. ORC-Cdc6 cooperative binding triggers Cdc6 ATPase activity. ORC ATPase activity soon follows and Cdc6 ATPase activity either directly or indirectly results in re-modeling of the complex accompanied by Mcm2-7 loading and DUP/Cdtl release (Fig. 2).
Mechanisms of Cyclin/Cdk regulation of the Pre-Replication Complex
Cyclin/Cdk regulation of the Pre-RC is varied and redundant and regulates nuclear compartmentalization, chromatin binding, catalytic activity and protein stability. In Xenopus and Drosophila, high Cyclin/Cdk activity inhibits ORC binding to chromatin (Findeisen et al., 1999; Remus et al., 2005). In mammals, Cdk phosphorylation of Cdc6 at N-terminal sites exposes a nuclear export signal leading to nuclear export in S phase
Figure 2. Replication initiation.
In mitotic and endocycles, Pre-Replication Complex assembly at origins of replication is restricted to G1 during a period of low Cyclin/Cdk (CDK) activity. Regulation of Pre-Replication Complex assembly is key to once-per-cell-cycle control of DNA replication. (A) Pre-Replication Complex assembly begins with cooperative ORC and Cdc6 binding to origin
DNA. DUP/Cdtl localizes after Cdc6. Sequential ATP hydrolysis by Cdc6, then ORC, leads to Mcm2-7 loading and completion of the Pre-Replication Complex (B). (C) At replication
initiation, McmlO localizes. S-phase kinases Dbf4/Cdc7 (DDK) and Cyclin/Cdk (CDK) are required for localization of Cdc45, which subsequently travels with the replication fork. Sld3 is required for Cdc45 localization during replication initiation.
A. Cdc6 and ORC cooperative binding
Cdc6 ATPase, ORC ATPase B. The Pre-Replication Complex
~KLJX~
DDK, CDK
C. Generation of the functional helicase
Duj
;/0,
(Delmolino et al., 2001). In addition, Cdc6 degradation is signaled by ubiquitylation by the SCF/Cdc4 E3 ligase in S. cerevisiae (Perkins et al., 2001) and in vitro experiments indicate that Cdc6 degradation occurs following Cyclin/Cdk phosphorylation of Cdc6 (Elsasser et al., 1999). Pre-RC assembly is also regulated by Cyclins binding to ORC and Cdc6. Clb5 binds to and inhibits Orc6 and Clb2 binds to and inhibits Cdc6 in S.
cerevisiae (Mimura et al., 2004; Wilmes et al., 2004).
Cyclin/Cdk regulation of DUP/Cdtl affects chromatin binding and protein stability. CyclinA/Cdk phosphorylation has been shown to inhibit chromatin binding of human DUP/Cdtl in vitro and inhibition of Cdkl activity in murine cells leads to
accumulation of dephosphorylated DUP/Cdtl onto chromatin (Sugimoto et al., 2004). In addition to chromatin localization, Cyclin/Cdk regulates DUP/Cdtl abundance.
CyclinA/Cdk2 phosphorylaton of human DUP/Cdtl has been shown to promote
DUP/Cdtl binding to the Skp2 F-box protein, a cofactor for the SCF E3 ligase, which has been shown to target DUP/Cdtl for proteolysis (Nishitani et al., 2006; Sugimoto et al., 2004). In Drosophila, consensus Cdk phosphorylation sites in the N-terminus of
DUP/Cdtl are required for cell cycle dependent degradation and DUP/Cdtl degradation appears to be dependent on CyclinE/Cdk2 phosphorylation (Thomer et al., 2004).
Cyclin/Cdk regulates chromatin binding of the MCM complex. Phosphorylation of Xenopus Mcm4 by CyclinB/Cdkl reduces its affinity for chromatin (Hendrickson et al,
1996). There is also evidence that Cdk2 phosphorylation of Mcm2 primes this subunit for phosphorylation by other kinases regulating chromatin association (Montagnoli et al., 2006).
The transition to replication
Following formation of the Pre-RC, the transition to replication requires the loading of multiple factors that unwind DNA and localize DNA polymerases (Fig. 2) (Pacek et al., 2006; Wohlschlegel et al., 2002). These include McmlO, Cdc45/Sld3, th GINS complex and Dpbl 1/Sld2. In Xenopus and S. cerevisiae, Mcml0O binds to origins in an Mcm2-7 dependent manner. Cdc45 localization, in turn, is dependent on Mcml0O (Sawyer et al., 2004; Wohlschlegel et al., 2002) and, in yeast, on Sld3 with which it forms a complex (Kamimura et al., 2001; Kanemaki and Labib, 2006; Nakajima and
Masukata, 2002). Two other protein complexes, localize to origins at replication. These are the GINS complex and Dpbl 1/Sld2. This assembly of complexes is required for recruiting DNA polymerases. Mcml0O and Cdc45 are primarily responsible for localization of DNApola-primase (Mimura et al., 2000; Ricke and Bielinsky, 2004; Uchiyama et al., 2001; Zou and Stillman, 2000). Recruitment of DNApolE to origins requires the GINS complex and Dpbl 1/Sld2 (Takayama et al., 2003).
Two S-phase kinases regulate replication initiation: Cyclin/Cdk and Dbf4/Cdc7. In Xenopus, Dbf4/Cdc7 associates with chromatin in an Mcm2-7 dependent manner prior to Cdc45 localization (Jares and Blow, 2000; Jares et al., 2004). Sequential kinase
activity appears to be important for DNA replication. In a Xenopus cell-free system, exposure of chromatin to Dbf4/Cdc7 and Cyclin/Cdk2 promoted efficient DNA replication but exposure of chromatin to these kinases in the reverse order did not (Walter, 2000). Cyclin/Cdk phosphorylation may promote initiation by promoting
Dpbl 1/Sld2 assembly. Mutation of all the potential Cdk phosphorylation sites of Sld2 has been shown to inhibit complex assembly and replication (Tak et al., 2006).
Replication Elongation
Several of the proteins involved in the transition to replication and in replication fork biogenesis travel with the replication fork. Mcml0, Cdc45, and the GINS complex have been identified at replication forks in Xenopus and S. cerevisiae (Aparicio et al.,
1997; Calzada et al., 2005; Gambus et al., 2006; Pacek et al., 2006). Mutants in S. cerevisiae McmlO0 and Cdc45 show stalled replication forks (Merchant et al., 1997; Tercero et al., 2000). Recently, a Cdc45/Mcm2-7/GINS (CMG) complex associated with helicase activity was purified from Drosophila extracts suggesting that this complex comprises the replication fork helicase (Moyer et al., 2006).
Three DNA polymerases localize to the replication fork: DNApola-primase, DNApol8 and DNApolE (Garg and Burgers, 2005). DNA pola-primase possesses RNA and DNA polymerase activities and mediates priming at initiation to start leading strand synthesis and travels with the replication fork to prime lagging strand synthesis.
DNApolI is thought to mediate Okazaki fragment maturation during lagging strand synthesis. DNApols is thought to perform leading strand synthesis. In addition, RPA, RFC, and PCNA are loaded. RPA consists of three subunits and is functionally
homologous to E. coli SSB and binds to single-stranded DNA. PCNA is a trimeric sliding clamp that increases the processivity of DNA polymerases. RFC loads PCNA.
Replication Fork Progression
Replication Fork Progression (RFP) is regulated by DNA secondary structure and chromatin bound proteins. Differences in Replication Fork Speed (RFS) have been observed for cells employing different modes of DNA replication. In Drosophila diploid cells, replication forks move at ~2.6 kb/min (Blumenthal et al., 1974). The RFS for Drosophila polytene larval salivary glands has been measured at ~300 bp/min
(Steinemann, 1981). During gene amplification, replication forks move at about ~50-100 bp/min (Spradling and Leys, 1988). These differences may be due to chromatin structure (for example, the persistence of cohesins on polytene chromosomes) or topological factors. Multiple, tandem replication forks during gene amplification may generate significant superhelical strain. At DAFC-66D, -~5 replication forks moving in the same direction are spaced -~10 kb apart.
RFS and RFP are also regulated by protein factors at the replication fork. Helicases are common targets for modulating RFP. Replication fork progression at normal speeds through S. cerevisiae telomeric and subtelomeric sequences requires the Rrm3p helicase, suggesting that cellular helicases have specialized functions (Ivessa et al., 2002). Not surprisingly, replication fork pausing mechanisms often target the helicase. During prokaryotic replication termination, the trans-acting factors Tus in E. coli and RTP in B. subtilis bind to and inhibit the replicative helicase (Bussiere and Bastia, 1999). In a proof-of-principle, a protein inhibitor of the replicative helicase was shown to reduce replication fork speed In E. coli, (Skarstad and Wold, 1995). Helicase
activity is regulated by Cyclin/Cdk phosphorylation (Ishimi et al., 2000), and this may be an important mechanism for regulating replication fork speed.
Regulation of replication fork progession may be important for gene expression. Polar intergenic Replication Fork Barriers (RFB), block replication fork progression opposing transcription at the rDNA locus of S. cerevisiae, S. pombe, mouse and Xenopus (Rothstein et al., 2000). In E. coli, head-on transcription severely inhibits replication fork progression while co-directional transcription does not, revealing the importance of coordinated transcription and replication (Mirkin and Mirkin, 2005). The interplay between DNA replication machinery and transcriptional machinery is not necessarily direct. Transcription factors may affect nucleosome arrangement or recruit chromatin re-modeling machinery (Bosco et al., 2001). During Drosophila follicle cell gene
amplification, mutants in transcription factors affect replication initiation (Beall et al., 2004; Beall et al., 2002; Bosco et al., 2001; Royzman et al., 1999). Some of these mutants may affect replication fork progression.
Summary
The mechanisms regulating DNA replication are conserved between mitotic cell cycles, endocycles and Drosophila follicle cell gene amplification. A period of low Cyclin/Cdk, a G1 phase, in which the Pre-RC can assemble is conserved between mitotic and endocycles. The Pre-RC is conserved during Drosophila follicle cell gene
amplification. Mechanisms for regulating replication initiation are conserved between different modes of DNA replication. Aspects of replication fork progression are likely to be conserved. Mcm2-7, Cdc45 and PCNA travel with the replication forks in mitotic cells
(Claycomb et al., 2002; Loebel et al., 2000). These proteins co-localize with replication forks during gene amplification. In addition, DUP/Cdtl, co-localizes with replication forks during gene amplification. It has been postulated that DUP/Cdtl is required at the replication forks to maintain Mcm2-7 at slow-moving replication forks (Claycomb et al., 2002).
We performed a screen to identify developmental regulators of mitotic and endocycles during Drosophila embryogenesis. Three cell cycle phenomena - cell cycle exit, mitotic cell cycles and endocycles - occur concurrently during Drosophila
embryogenesis. We took advantage of this period to screen for developmental regulators of S-phase using PCNA, as an S-phase marker. We identified a class of mutants that displayed large nuclei in normally diploid tissues. In this class, we identified new mutations in pavarotti and tumbleweed, pav3C53 and tum32a-20, which are required for
cytokinesis. In addition, we characterized a new cyclinE mutant, cyclinElf36, displaying
increased replication fork progression during gene amplification. This previously undescribed gene amplification defect implicates CyclinE in regulating replication fork progression and raises the intriguing possibility that replication fork progression is plastic during gene amplification and may be subject to developmental regulation.
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CHAPTER Two
New mutants affecting developmentally regulated cell cycles during
Drosophila embryogenesis
Eugenia A. Park and Terry L. Orr-Weaver
Whitehead Institute and Dept. of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142
SUMMARY
During Drosophila embryogenesis, cell cycle exit, mitotic cell cycles and endocycles all occur within a narrow time span. The developmental patterning of these cell cycles are well characterized and afford a unique opportunity to study developmental regulation of the cell cycle. We screened 300 EMS mutants for developmentally uncoordinated
replication by an in situ hybridization assay for PCNA transcription, a marker of the G1/S transition. We identified 30 mutants that may reflect functions in developmental
signaling, G1/S transcription and DNA replication. We further characterized a class of mutants displaying large, apparently polyploid nuclei in normally diploid cells. 3C157, Ir8 and 2k32 - of which 3C157 and Ir8 are allelic - displayed large and diffuse DNA masses in isolated cells of the nervous system. 32a-20 (formerly 32a) and 3C53 displayed large nuclei in the nervous system and epidermis. We cloned these mutants and identified new alleles of tumbleweed, tum3 2
a-20, and pavarotti, pavC 533 . These mutants have defects consistent with blocks to cytokinesis.
INTRODUCTION
In metazoans, divergent cell cycles must be regulated accurately throughout development to build and maintain a viable organism. The G1/S/G2/M cell cycle is only one of multiple cell cycles. Variants of the archetypal cell cycle are utilized in different developmental contexts to achieve different aims. The early embryonic divisions of insects, marine invertebrates and amphibians are rapid S/M cycles. These cycles allow for speedy embryogenesis, potentially important for organisms with exposed and vulnerable embryos. Endocycles are variants of the archetypal cell cycle that lack complete mitoses but consist of discrete S and G1 phases with one round of DNA replication occurring per endocycle (Smith and Orr-Weaver, 1991). These cycles generate polyploidy and are thought to be a strategy for increasing growth and metabolic capacity without the large scale cytoskeletal rearrangements required by mitosis (Edgar and Orr-Weaver, 2001).
A key requirement for all of these cell cycles is the restriction of Cyclin/Cdk activity. A window of low Cyclin/Cdk activity is required for assembly of pre-replication complexes at replication origins (Hua et al., 1997). During G1/S/G2/M cycles,
Cyclin/Cdk activity remains high throughout the cell cycles except for a window in early G1 following downregulation of Cyclins A and B and preceding upregulation of CyclinE. During the early embryonic S/M cycles of Xenopus, nuclear compartmentalization
restricts Cyclin/Cdk activity (Blow and Laskey, 1988). Some endocycling tissues show vestiges of mitosis ranging from chromosome condensation to nuclear envelope
breakdown (Edgar and Orr-Weaver, 2001) and utilize mitotic machinery to achieve low Cyclin/Cdk activity. Mutations in morula, an APC subunit, lead to ectopic spindle formation and chromosome condensation in Drosophila nurse cells suggesting that
regulators of mitosis are expressed at low levels in these cells (Kashevsky et al., 2002; Reed and Orr-Weaver, 1997). To modulate activity, Cyclin/Cdk complexes are regulated on many levels including oscillatory Cyclin expression, regulatory phosphorylation, inhibition by Cyclin/Cdk inhibitors (CKI), and through targeted degradation of Cyclins.
Developmental signaling plays a key role in regulating different cell cycles. In Drosophila, Notch signaling mediates a mitotic to endocycle switch in ovarian follicle cells, and no less than 36 pattern formation genes are involved in the developmental regulation of mitotic cell cycles during embryogenesis (Deng et al., 2001; Edgar et al.,
1994; Keller Larkin et al., 1999; Schaeffer et al., 2004; Shcherbata et al., 2004).
Drosophila embryogenesis provides an elegant example of developmental regulation of the cell cycle. Cell cycle exit and three different cell cycles -S/M, S/G2/M and S/G -occur dynamically in a 6 hour time span (Fig. 1). Cycles 1-13 consist of rapidly alternating S and M phases and are nuclear divisions that occur more or less
synchronously in a common cytoplasm. Cycles 14-16, the postblastoderm divisions, consist of S, G2 and M phases. These divisions (S/G2/M) occur in mitotic domains in which cells differentiating into the same tissue undergo mitosis synchronously and at the same developmental time (Foe et al., 1993). Following cycle 16, the embryonic epidermis exits the cell cycle while cells of the developing nervous system continue to undergo mitotic cycles. Also following cycle 16, the developing larval tissues initiate endocycles consisting of S and G phases and continue these through embryogenesis and larval development resulting in highly polytene tissues. Embryonic endocycles occur in spatiotemporal domains reminiscent of the mitotic domains of the postblastoderm divisions (Fig. 2) (Smith and Orr-Weaver, 1991).
Figure 1. Diagram of variant cell cycles utilized during embryogenesis.
Following fertilization, cell cycles 1-13 are syncitial divisions that occur synchronously in a common cytoplasm. These rapid S/M cycles correspond to stages 1-8 of embryonic development. By cycle 14, cellularization is complete and a gap phase is added
coincident with a requirement for zygotic string/CDC25 transcription. Cycles 14-16 (the postblastoderm divisions) consist of S/G2/M phases and occur in mitotic domains in which groups of cells differentiating together undergo mitoses at different times. The dorsal epidermis undergoes cycles 14, 15, and 16 in stages 9, 10 and 11 respectively. Following cycle 16, the epidermis exits the cell cycle in stages 11 and 12 (not shown). The developing nervous system continues to cycle mitotically while the endodomains, or developing larval tissues, initiate S/G endocycles in stage 11. Shown in the image is a stage 12 embryo (anterior is to the left) in situ hybridized with antisense riboprobe against PCNA, which is expressed at G1/S, to visualize the nervous system (NS) and the endodomains (ENDO).
