Thesis
Reference
TopBP1 functions with 53BP1 in the G1 DNA damage response
CESCUTTI, Rachele
Abstract
TopBP1 (Topoisomerase II β binding protein) est une protéine qui sert de point de contrôle du cycle cellulaire et qui colocalise avec ATR (Ataxia telangiectasia and Rad3 related) aux sites de stress réplicatif. Nous démontrons ici que TopBP1 colocalise également avec 53BP1 (p53 binding protein 1) aux sites de cassure double brin, mais seulement lors de la phase G1 du cycle cellulaire. Le recrutement de TopBP1 aux sites de stress réplicatif est dépendant des domaines 1 et 2 plus 7 et 8 de BRCT, tandis que le recrutement aux sites de cassure double brin est dépendant des domaines 1 et 2 plus 4 et 5 de BRCT. Les domaines 4 et 5 de BRCT interagissent avec 53BP1, qui est nécessaire pour le recrutement de TopBP1 aux sites de cassure double brin en phase G1 du cycle cellulaire. Comme TopB1 contient un domaine important pour l'activation d'ATR, nous avons testé s'il contribuait au point de contrôle G1 du cycle cellulaire. En irradiant des cellules en phase G1, puis en contrôlant leur entrée en phase S, nous avons observé un défaut de ce point de contrôle après déplétion de TopBP1, 53BP1 ou ATM à l'aide de [...]
CESCUTTI, Rachele. TopBP1 functions with 53BP1 in the G1 DNA damage response. Thèse de doctorat : Univ. Genève, 2011, no. Sc. 4309
URN : urn:nbn:ch:unige-158875
DOI : 10.13097/archive-ouverte/unige:15887
Available at:
http://archive-ouverte.unige.ch/unige:15887
Disclaimer: layout of this document may differ from the published version.
Département de Biologie Moléculaire
____________________________________________________________
TopBP1 Functions with 53BP1 in the G1 DNA Damage Response
THÈSE
Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Rachele Cescutti
de Monza (Italie)
Thèse N° 4309
GENÈVE
Atelier de reproduction de UniMail 2011
I would like to thank my thesis advisor, Prof. Thanos Halazonetis, for his mentorship during my graduate career.
I also thank the past and present members of the lab in particular Simona Negrini and Masaoki Kohzaki, the department of Molecular Biology at the University of Geneva and the members of my thesis committee: Prof. Stéphan Thore and Dr.
Manuel Stucki.
TopBP1 functions with 53BP1 in the G1 DNA damage response
TopBP1 (Topoisomerase II β Binding Protein 1) is a checkpoint protein that
colocalizes with ATR at sites of DNA replication stress. We show here that TopBP1 also colocalizes with 53BP1 (p53 Binding Protein 1) at sites of DNA Double Strands Breaks (DSBs), but only in the G1 phase of the cell cycle. Recruitment of TopBP1 to sites of DNA replication stress was dependent on BRCT domains 1 and 2 plus 7 and 8, whereas recruitment to sites of DNA DSBs was dependent on BRCT domains 1 and 2 plus 4 and 5.
BRCT domains 4 and 5 interacted with 53BP1 and recruitment of TopBP1 to sites of DNA DSBs in G1 was dependent on 53BP1.
Since TopBP1 contains a domain important for ATR activation, we examined whether it contributes to the G1 cell cycle checkpoint. By monitoring entry of irradiated G1 cells into S phase, we observed a checkpoint defect after siRNA- mediated depletion of TopBP1, 53BP1 or ATM. Thus, TopBP1 may mediate the checkpoint function of 53BP1 in G1.
TopB1 interragit avec 53BP1 dans la réponse au dommage à l’ADN en phase G1
TopBP1 (Topoisomerase II β binding protein) est une protéine qui sert de point de contrôle du cycle cellulaire et qui colocalise avec ATR (Ataxia telangiectasia and Rad3 related) aux sites de stress réplicatif. Nous démontrons ici que TopBP1 colocalise également avec 53BP1 (p53 binding protein 1) aux sites de cassure double brin, mais seulement lors de la phase G1 du cycle cellulaire. Le recrutement de TopBP1 aux sites de stress réplicatif est dépendant des domaines 1 et 2 plus 7 et 8 de BRCT, tandis que le recrutement aux sites de cassure double brin est dépendant des domaines 1 et 2 plus 4 et 5 de BRCT. Les domaines 4 et 5 de BRCT interagissent avec 53BP1, qui est nécessaire pour le recrutement de TopBP1 aux sites de cassure double brin en phase G1 du cycle cellulaire. Comme TopB1 contient un domaine important pour l’activation d’ATR, nous avons testé s’il contribuait au point de contrôle G1 du cycle cellulaire. En irradiant des cellules en phase G1, puis en contrôlant leur entrée en phase S, nous avons observé un défaut de ce point de contrôle après déplétion de TopBP1, 53BP1 ou ATM à l’aide de petits ARN interférents. TopBP1 pourrait donc servir d’intermédiaire pour la fonction de point de contrôle du cycle cellulaire de 53BP1.
AKNOWLEDGEMENTS ii
ABSTRACT iii
FRENCH ABSTRACT iv
TABLE OF CONTENTS v
TABLE OF ABBREVIATIONS viii
TABLE OF FIGURES x
CHAPTER 1: INTRODUCTION 1
1.1 DNA damage response 1
1.2 Sensor 3
1.2.1 ATM 3
1.2.2 MRN complex 6
1.2.3 ATR 9
1.2.4 RAD17/RFC and 9-1-1 complex 12
1.3 Mediators 13
1.3.1 53BP1 (p53 binding protein 1) 14
1.3.2 TopBP1 (Topoisomerase II β binding protein 1) 17 1.3.3 MDC1 (Mediator of DNA damage checkpoint 1) 21
1.3.4 Claspin 23
1.3.5 RNF8 (RING finger 8) 23
1.4 Transducers 25
1.5 Effectors 26
1.5.1 CDC25 family 26
1.5.2 p53 27
1.6 The G1 checkpoint 28
1.7 The intra-S-checkpoint 28
1.8 The G2 checkpoint 29
CHAPTER 2: MATERIAL AND METHODS 33
2.1 Cell lines 33
2.2 Recombinant plasmids and antibodies 33
2.3 Focus formation assay and immunofluoresce 34 2.4 Small interfering RNA (siRNA) Transfection 35 2.5 Preparation of the cell extracts, immunoblotting and coprecipitation assays
36
2.6 Checkpoint assay and facs analysis 36
Protocol N°1: Immunofluorescence 38
Protocol N°2: DNA transfection or siRNA Transfection 40
Protocol N°3: whole cell extract 42
Protocol N°4: Nuclear extraction 43
Protocol N°5: Preparation of IgG dynabeads 45
Protocol N°6: FACS 47
Protocol N°7 Stable cell line 49
CHAPTER 3: RESULTS 50 3.1 TopBP1 colocalizes with either RPA or 53BP1 50
3.2 TopBP1 colocalization is cell cycle regulated 50 3.3 Mapping the domains that mediateTopBP1recruitment 51 3.4 Different pairs of TopBP1 BRCT domains have distinct properties for
recruitment to site of DNA damage 52
3.5 TopBP1 recruitment to sites of DNA DSBs is 53BP1-dependent and ATM-
dependent 54
3.6 TopBP1 and 53BP1 are required for the G1 DNA damage checkpoint 55
CHAPTER 4: DISCUSSION 75
REFERENCES 85
TABLE OF ABBREVIATIONS
DNA DSBs DNA Double Strands Breaks DDR DNA Damage Response
PIKK Phosphotidylinositol 3-Kinase-like Kinase A-T Ataxia-Telangiectesia
ssDNA Single Stranded DNA CDK Cyclin Dependent Kinases DDK Dependent Kinases
GFP Green Fluorescent Protein MEF Mouse Embryos Fibroblasts
HU Hydroxyurea
U2OS Human Osteosarcoma cell line
EdU Ethynyl Deoxyuridine
BrdU Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) DAPI 4',6-diamidino-2-phenylindole
FACS Fluorescence-activated cell sorting IF Immunofluoresce
Co-IP Co-Immunoprecipation IR Irradiation
UV Ultra-violet light
AAD ATR Activation Domain
NHF Normal Human Fibroblasts A-TF Ataxia-Telangiectasia Fibroblasts
Figure 1 31
Figure 2 32
Figure 3.1. Endogenous TopBP1 colocalizes with either RPA or 53BP1 in irradiated cells depending on the phase of the cell cycle 58 Figure 3.2. Mapping the domains that mediate recruitment of TopBP1 to IR-induced
53BP1 or RPA foci 59
Figure 3.3. Different pairs of TopBP1 BRCT domains have distinct properties for
recruitment to sites of DNA damage 60
Figure 3.4. TopBP1 recruitment to sites of DNA DSBs is 53BP1- and ATM-
dependent 62
Figure 3.5. TopBP1 and 53BP1 are required for the G1 DNA damage checkpoint 64
Suppl. Figure 1 65
Suppl. Figure 2 Cell cycle dependency of IR-induced TopBP1 foci 66 Suppl. Figure 3 Mapping the domains that mediate recruitment of TopBP1 to IR-
induced 53BP1 or RPA foci 67
Suppl. Figure 4 Different pairs of TopBP1 BRCT domains have distinct properties
for recruitment to sites of DNA damage 68
Suppl. Figure 5 69
recruitment to DNA damage sites 70
Suppl. Figure 7 72
Suppl. Figure 8 Analysis of the histone H2AX-, MDC1- and RNF8-dependency of
TopBP1 recruitment to DNA damage sites 73
Suppl. Figure 9 Model for the checkpoint function of 53BP1 and TopBP1 at sites of
DNA DSBs in G1 cells 74
CHAPTER 1
INTRODUCTION
1.1 DNA damage response
Growth and division of a single cell to yield two daughters cells requires the coordination of numerous events, in particular the faithful replication and partitioning of the cell’s genetic material to each daughter cell. Errors in this process could threaten cell survival leading to immunodeficiency, cancer, pathology sequelae and even death (Abraham 2001; Nyberg et al. 2002; Sancar et al. 2004).
To ensure the fidelity of cell division, evolution has overlaid general mechanisms called checkpoints that monitor the successful completion of cell cycle events.
Checkpoints are typically dispensable for cell cycle events per se, but rather they assure that events are completed correctly and in the proper order.
When one cell cycle event, has not been successfully completed, checkpoint activation slows down or arrests cell cycle progression, thereby allowing time for appropriate repair mechanisms to correct the genetic lesions before they are passed to the next generation of daughter cells.
Given that genomes of eukaryotic cells are constantly under assault by environmental agents (e.g., UV light and reactive chemicals) as well as by byproducts of normal intracellular metabolism (e.g., reactive oxygen intermediates
and inaccurately replicated DNA), maintaining a complete, undamaged genome is a major challenge (Figure 1)
All eukaryotic cells have four phases within the cell cycle, G1, S, G2 and M and all the transition steps from one phase to the next one, are tightly controlled to minimize the accumulation of damage.
Checkpoint systems represent sophisticated machinery which relies on the interplay of several proteins that act in concert to translate the signal of damaged DNA into responses of cell cycle arrest and repair.
These proteins can be classified in three different groups a) SENSOR proteins that recognize damaged DNA and function to signal the presence of abnormatilities, initiating a biochemical cascade of activity; b) TRANSDUCER proteins that amplify the damage signal, phosphorylating targets proteins; and C) EFFECTOR proteins, that include the most downstream targets of the transducer protein kinases, to prevent cell cycle progression (Abraham 2001; Nyberg et al. 2002; Sancar et al.
2004).
Notably, there is not an absolute demarcation between the various components of the cell cycle checkpoints and recently a fourth class of checkpoint proteins, called MEDIATORS, has been identified. These proteins are placed between sensor and transducers.
1.2 Sensors
DNA Damage Response (DDR) requires the recognition of DNA damage to initiate the subsequent cascade.
Two groups of proteins have been identified as checkpoint-specific damage sensors:
the two phosphotidylinositol 3-kinase-like kinase (PIKK) family members, ATM and ATR, and protein, such as the MRE11-Rad51-Nbs1 and the RFC/PCNA (clamp loader/polymerase clamp)-related Rad17-RFC/9-1-1 complex that help activate ATM and ATR, respectively (Durocher and Jackson 2001; Melo and Toczyski 2002).
ATM and ATR, also act as transducers in the DDR pathways, thus their classification as sensors is still debated.
1.2.1 ATM
ATM (Ataxia, Telangiectesia Mutated) is one of the master controllers of the DNA damage response. Mutations in ATM cause ataxia-telangiectesia (A-T) in humans, a condition primarily characterized by cerebellar degeneration, immunodeficiency, genome instability, clinical radiosensitivity and cancer predisposition (Sancar et al. 2004).
As mentioned above, ATM belongs to the phosphatidylinositol 3-kinase-like kinase family which in mammals comprises ATM, ATR, ATX/SMG-1, DNA-PK and
mTOR/FRAP. All members, except mTOR, have a role in the DDR. The mTOR protein is involved in regulating cell growth in response to nutrient levels and mitogenic stimuli (Proud 2002).
ATM is a 350 KDa oligomeric protein. It shares conserved domains with PIKK family members: the FRAP/ATM/TRRAP (FAT) domain, the FAT-C terminal (FATC) domain and the kinase domain.
The functions of the FAT domain and the C-terminal FAT-C domain have to be elucidated. Most likely are involved in regulating ATM kinase activity. The kinase domain contains the catalytic domain site that recognizes and phosphorylates substrates at SQ/TQ site (serine or threonine followed by a glutamine) (Shiloh 2003).
Additionally the N-terminus of ATM possesses many HEAT (Huntingtin, Elongation factor 3, A subunit of PP2A, and TOR1) repeats. These motifs seem to be important for protein-protein interactions but their function in ATM is still poorly understood (Falck et al. 2005; You et al. 2005).
ATM activation. ATM mainly responds to DNA Double Strands Breaks (DSBs) by phosphorylating numerous substrates (Shiloh 2003). Indeed, its kinase activity strongly increases in response to DNA damage (Banin et al. 1998; Canman et al. 1998).
ATM-mediated phosphorylation either enhances or represses the activity of its targets, thereby promoting cell cycle arrest and DNA repair.
Following DNA damage, ATM is rapidly recruited to the sites of DNA DSBs. This rapid recruitment is visualized by immunofluorescence monitoring retention of ATM at chromatin and by the chromatin immunoprecipitation (Chip) assay (Dehreimer and Kastan 2010).
ATM exists in an inactive dimer in undamaged cells, but after induction of DNA damage or treatment with agents that alter chromatin structure, ATM undergoes an intramolecular auto-phosphorylation on serine 1981 resulting in disassociation of the dimer into active monomers (Bakkenist and Kastan 2003).
When ATM becomes active, it regulates by phosphorylation several proteins within the same pathway. In regulating p53, ATM employs three different means to stabilize the protein. First, ATM phosphorylates p53 on Ser 15 and this contributes primarily to enhancing the activity of p53 as a transcription factor (Canman et al.
1998). Second, the phosphorylation of Ser 20 of p53 by Chk2 acting downstream of ATM reduces the ability of ubiquitin ligase Mdm2 to bind p53, thus promoting p53 stabilization (Haupt et al. 1997; Chehab et al. 2000; Hirao et al. 2000; Shieh et al.
2000). Finally, ATM ensures stabilization of p53 by phosphorylating directly Mdm2, (Khosravi et al. 1999).
Another protein activated by ATM is BRCA1 (Breast-Ovarian Cancer Susceptibility 1). It is phosphorylated on Ser 1387 during the intra-S checkpoint (Xu et al. 2002), whereas its phosphorylation on Ser 1423 spurs its involvement in the G2-M checkpoint (Xu et al. 2001). So the phophorylation on different sites might regulate its activity in different pathways. Moreover, activated Chk2, downstream ATM,
additionally phosphorylates BRCA1 on another site that is required for the dissociation of BRCA1 and Chk2 (Lee et al. 2000). Finally, ATM also phosphorylates CtiP, forcing its dissociation from BRCA1 (Li et al. 2000b). Ctip then can function with the MRN complex promoting DNA end resection at sites of DNA DSB (Sartori et al. 2007).
Other important substrate of ATM is the histone protein H2AX. H2AX is phosphorylated at C-terminal SQ motif (Ser 139). Phosphorylated H2AX is detected by immunuofluorescence through the formation of distinct foci immediately after the induction of DNA damage (Rogakou and Sekeri-Pataryas 1999; Sedelnikova et al.
2002; Kinner et al. 2008) and this process serves as rapid and powerful mechanism for amplifying the damage signal via recruitment of mediator and transducer proteins (Shiloh 2003; Kinner et al. 2008).
1.2.2 MRN complex
In addition to ATM, the MRN complex, which is highly conserved throughout all kingdoms of life, is constituted by Mre11-Rad50-Nbs1. It plays an important role in DSBs repair and it is rapidly recruited at the DNA damage sites (Lavin 2007). The MRN complex is known as sensor of DNA DSBs and it does not need ATM for its recruitment to the DNA damage sites. Furthermore, recent studies performed in cells lacking Mre11 or Nbs1, have demonstrated a requirement of the
MRN complex for a full ATM activation. Moreover, once ATM is activated, the MRN complex becomes one of its substrates. It has also been shown that ATM interacts physically with Nbs1 via its HEAT repeats (Falck et al. 2005).
Taken together these data, demonstrate that ATM and the MRN complex work in concert with one another at the sites of DNA DSBs to promote an effective DDR and repair.
MR11. MRN complex possesses exonuclease and endonuclease functions mediated by Mre11 that are important for DSB repair and homologous recombination, as well as signaling funtions (Assenmacher and Hopfner 2004).
Mre11 functions as a dimer and interacts with Rad50 (Hopfner et al. 2001; Williams et al. 2008). Mre11 exhibits 3’-5’ double-strand exonuclease and single- and double strand endonuclese activities (Trujillo et al. 1998; Paull and Gellert 1999). In some systems, the nuclease activity of the Mre11 is dependent on the presence of Nbs1, ATP and manganese (Paull and Gellert 1999).
Rad50. It belongs to the structural maintenance of chromosome (SMC) family proteins, involved in chromosome condensation and sister chromatid cohesion in eukaryotes (Hirano 2002). The N-terminal and the C-terminal domains contain Walker A and Walker B motifs required for ATPase activity, as well as being responsible for DNA binding and unwinding. Outside of the Walker A and B motifs, the central region of Rad50 is composed of a large coiled-coil structure that can fold back on itself, via a “hinge” region (Hopfner et al. 2001). Notably, ATP activity
seems to be critical for Rad50 function, which is believed to be DNA end binding and tethering at the site of break in order to facilitate repair (Alani et al. 1989; de Jager et al. 2001).
Nbs1. The name Nbs1 refers to Nijmegen Breakage Syndrome, which shares a similar phenotype with ataxia-telangiectasia (observed in patient with mutant ATM), and ataxia-telangiectasia-like disorder (observed in patient with mutant Mre11).
Nbs1 is a protein of 754 aminoacids and apparently does not have any known enzymatic activities, but it clearly regulates MRN funtions. First, it is essential for the nuclear localization of Mre11 and Rad50 (Carney et al. 1998; Cerosaletti et al.
2000; Desai-Mehta et al. 2001; Shima et al. 2005; Tsukamoto et al. 2005). Second, it is needed for the rapid assembly of MRN complexes at sites of DNA damage (Kobayashi et al. 2002; Horejsi et al. 2004; Lee and Paull 2004). Third, Nbs1 stimulates the activities of Rad50 and Mre11 as it is required for nucleotide- dependent DNA binding by the MRN complex and ATP-dependent DNA unwinding (Paull and Gellert 1999; Lee and Paull 2004).
Nbs1 contains at its N-terminal end a forkhead-associated (FHA) domain followed by two tandemly repeated BRCA1 carboxyl-terminus BRCT motifs (Bork et al.
1997; Becker et al. 2006). Both domains, the FHA and tandem BRCT domains, have been shown to have specificity for phospho-peptides and thus, mediate interactions with phosphorylated DDR proteins (Hofmann and Bucher 1995; Bork et al. 1997;
Durocher et al. 1999; Li et al. 2000a; Yu et al. 2003; Mahajan et al. 2008).
The extreme carboxyl-terminus of Nbs1 contains short motifs responsible for its interactions with Mre11 and ATM (Carney et al. 1998; Matsuura et al. 1998; Varon et al. 1998; Desai-Mehta et al. 2001; Falck et al. 2005; Chen et al. 2008).
1.2.3 ATR
ATR was discovered in the human genome database as a gene with sequence homology to ATM and SpRad3, hence the name ATR (ATM and Rad 3 related) (Cimprich et al. 1996). The gene encodes a protein of 303 KDa with a C-terminal kinase domain and regions of homology to other PIKK family members. Knockout of ATR in mice results in embryonic lethality (Brown and Baltimore 2000; de Klein et al. 2000) and mutations causing partial loss of ATR activity in humans have been associated with the human autosomal recessive disorder Seckel syndrome, which shares features in common with A-T (O'Driscoll et al. 2003).
ATR, like ATM, is a protein kinase with specificity for SQ/TQ sites and as a consequence the function of ATR is partly redundant with that of ATM. In fact substrates of ATR and ATM are partially overlapping (Poehlmann and Roessner 2010).
ATR activation. ATR-activating DNA lesions have in common the ability to expose single-stranded DNA (ssDNA), often as a consequence of stalling the replicative polymerases. The relative insensitivity of the replicative helicase to these lesions
causes an uncoupling of polymerase and helicase activities resulting in ssDNA gaps (Costanzo and Gautier 2003; Byun et al. 2005). The ssDNA is a common ATR- activating signal (Costanzo and Gautier 2003; Zou and Elledge 2003).
ssDNA is not naked but it is rapidly coated by the ssDNA binding protein RPA (Fanning et al. 2006). ATR recognition of RPA-ssDNA depends on another protein, ATR-interacting protein (ATRIP) (Cortez et al. 2001). The stabilities of ATR and ATRIP are linked and these two proteins interact constitutively. There are no known differences in the phenotypes that result from the loss of ATR or ATRIP in any organism, suggesting that ATRIP can be considered an essential subunit of ATR.
Biochemical studies indicate that ATRIP binds RPA directly through an evolutionarily conserved binding surface (Ball et al. 2007). The primary interaction involves an α-helix in ATRIP that binds to the N-terminus of the RPA70, the large subunit of RPA (Ball et al. 2007).
Although RPA-ssDNA might be sufficient for localization of ATR-ATRIP complex to sites of DNA replication stress, it is not enough for ATR activation (Stokes et al.
2002; MacDougall et al. 2007). Colocalization of the Rad9-Rad1-Hus1 (9-1-1) complex to ssDNA is also required. The 9-1-1 heterotrimeric ring is similar to the replicative sliding clamp PCNA (Ellison and Stillman 2003; Zou et al. 2003) and it is loaded onto primer-template junctions in an ATP-dependent reaction that involves the Rad17-RFC2-5 clamp loader (Bermudez et al. 2003; Ellison and Stillman 2003;
Zou et al. 2003). The 9-1-1 complex recognizes the free 5’ DNA end that is adjacent to a stretch of RPA-ssDNA (Ellison and Stillman 2003; MacDougall et al. 2007) and this 5’ end appears to be relevant for activation of the checkpoint.
Another important protein implicated in ATR activation is TopBP1 (Topoisomerase II β Binding Protein) (Yamane et al. 1997; Garcia et al. 2005; Kumagai et al. 2006;
Lee et al. 2007; Mordes et al. 2008) which has been recently found to interact with 9-1-1 and Rad17 in a tripartite complex upon blockage of DNA replication (Lee and Dunphy ; Furuya et al. 2004; Delacroix et al. 2007; Lee et al. 2007; Yan and Michael 2009).
TopBP1 contains eight BRCA1 C-terminus (BRCT) repeats (Garcia et al. 2005).
Located between BRCT domains 6 and 7 of TopBP1, is a recently defined ATR activation domain (AAD). TopBP1 uses this domain to interact with DNA-bound ATR-ATRIP, and this interaction stimulates ATR kinase activity (Kumagai et al.
2006; Lee et al. 2007; Mordes et al. 2008; wanMordes et al. 2008).
Once ATR is activated, it phosphorylates key substrates such as the kinase Chk1 which becomes phosphorylated on Ser 317 and on Ser 345 (sakWalworth and Bernards 1996; Lopez-Girona et al. 2001). When Chk1 is phosphorylated, it is released from chromatin and it phosphorylates several other substrates (Smits et al.
2006). Key Chk1 targets for controlling cell cycle transitions are the CDC25 phosphatases (Boutros et al. 2006). Human cells have three CDC25 proteins that regulate cell-cycle transitions by removing the inhibitory phosphorylation of Cyclin- Dependent Kinases (CDKs).
Chk1 phosphorylation of CDC25 proteins inhibits their activity and prevents CDK activation (Furnari et al. 1997; Sanchez et al. 1997). This is the major checkpoint mechanism that prevents entry in mitosis.
ATR-signaling through Chk1 is also crucial for regulating replication: it slows down DNA replication by inhibiting origin firing.
Chk1 phosphorylation by ATR is regulated by a mediator protein named Claspin which brings ATR and Chk1 together (Kumagai and Dunphy 2000; Kumagai and Dunphy 2003).
Rad17 is also phoshorylated by ATR, and this promotes the interaction between Claspin and Rad17, which might be another mechanism for signal amplification (Bao et al. 2001; Wang et al. 2006).
A final class of ATR substrates comprises those that regulate DNA repair like BRCA1, Warner syndrome ATP-dependent helicase (WRN), Bloom syndrome protein (BLM) (Tibbetts et al. 2000; Davies et al. 2004) and Fanconi-Anemia protein FANCD2 (Andreassen et al. 2004).
1.2.4 RAD17/RFC and 9-1-1 complex
These two protein complexes function together in the DNA replication checkpoint. The Rad17/RFC complex is a checkpoint specific structural homolog of the replication factor RFC. The replicative form of RFC is a heteropentamer composed of p140, p40, p38, p37 and p36 and it acts as clamp loader of PCNA (Bell and Dutta 2002). In Rad17/RFC, the p140 subunit is replaced by the 75 kDa Rad17 protein (Griffiths et al. 1995; Griffith et al. 2002) and interacts with the four small
RFC subunits. The Rad17/RFC clamp loader loads the 9-1-1 complex onto DNA at the sites of DNA damage in an ATP-dependent manner.
The heterotrimeric complex 9-1-1, composed of three subunits Rad9, Hus1 and Rad1, is loaded at 5’ ssDNA/dsDNA junctions generated at the damage sites.
The C-terminus of Rad9 is constitutively phosphorylated on Ser 387 during cell cycle progression (St Onge et al. 2003) and this allows interaction with TopBP1 via its BRCT domain 1 and 2 during DNA replication stress (Makiniemi et al. 2001;
Delacroix et al. 2007). The binding between the 9-1-1 complex and TopBP1 is necessary for Chk1 phosphorylation by ATR (Delacroix et al. 2007) and it has been proposed that the 9-1-1 complex recruits TopBP1 at the DNA damage sites where it can enhance ATR activity by its Activation Domain (AAD) (Kumagai et al. 2006).
1.3 Mediators
These proteins simultaneously associate with DNA damage sensors and signal transducers at certain phases of the cell cycle and as a consequence help provide signal transduction specificity.
In humans, proteins that contain the BRCT protein-protein interaction module fit into this mediator category: the p53 Binding Protein (53BP1) (Schultz et al. 2000;
Wang et al. 2002), Topoisomerase II β Binding Protein 1 (TopBP1) (Yamane et al.
2002), the Mediator of DNA Checkpoint 1 (MDC1) (Goldberg et al. 2003; Stewart
et al. 2003), the Breast- Ovarian Cancer Susceptibility Protein1 (BRCA1) (Hashizume et al. 2001).
Other proteins classified as mediators are Claspin and the E3 ubiquitin ligase RNF8 (Xu and Stern 2003; Mailand et al. 2007)
Even for this class of proteins, the demarcation line is not so strict.
1.3.1 53BP1 (p53 Binding Protein 1)
53BP1 was originally identified in a yeast two hybrid screen using p53 as bait (Iwabuchi et al. 1994). It was found to bind to the central DNA binding region of p53, frequently mutated in tumors (Vogelstein et al. 2000).
The 53BP1 gene maps to chromosome 15q15-21 and encodes a protein product of 1972 amino acids residues.
53BP1 has a tandem Tudor domain and two C-terminal BRCT repeats , the latter ones are highly homologous to those in S. Cerevisiae RAD9 and S. Pombe CRB2 and constitute residues 1714-1850 and 1865-1972 of the protein (Huyen et al. 2004;
Alpha-Bazin et al. 2005).
Owing to the high homology with DNA damage checkpoint proteins S.Cerevisiae Rad9 and S. Pombe Crb2, a role of 53BP1 in the DNA DSBs response was first proposed by Halazonetis and colleagues (Schultz et al. 2000). 53BP1 rapidly localizes to discrete nuclear foci after exposure to ionizing radiation (Anderson et al.
2001; Adams and Carpenter 2006). These foci represent sites of DNA double strand
breaks, as they colocalize with DSB markers such γH2AX or MRN complex (Schultz et al. 2000; Xia et al. 2001).
53BP1 foci are present as early as 5 minutes after exposure to as low as 0.5 Gy ionizing radiation. The maximum number of foci is reached within 15-30 minutes post radiation, and the number steadily declines to baseline levels after 16 hours (Schultz et al. 2000).
The minimal region of 53BP1 required for focal recruitment has been mapped to amino acids 1220-1711, encompassing the Tudor domain (amino acids 1486-1602) (Iwabuchi et al. 1994; Huyen et al. 2004) and a region required for 53BP1 oligomerization (Zgheib et al. 2009). The BRCT domains and the N-terminal of 53BP1 are not essential for focal recruitment (Morales et al. 2003).
Efficient recruitment of 53BP1 depends on a number of upstream factors, including phosphorylation of H2AX at Ser 139 (Celeste et al. 2003), recruitment of both MDC1 (Stewart et al. 2003; Bekker-Jensen et al. 2005) and E3 ubiquitin ligase RNF8 (Huen et al. 2007; Mailand et al. 2007), methylation of H3 and H4 (Huyen et al. 2004; Botuyan et al. 2006) and Tip60 HAT (histone acetyltransferase) activity (Murr et al. 2006).
In response to DNA DSBs induction, H2AX is phosphorylated by the PIKKs to form γH2AX (Rogakou et al. 1999). MDC1 then binds to this modified histone residue via its BRCT domains. MDC1, once phosphorylated in an ATM dependent- manner, recruits RNF8 at the DNA damage sites (Huen et al. 2007; Mailand et al.
2007), which then catalyzes one or more ubiquitylation events of H2AX (Ikura et al.
2007). All these mechanisms are essential for 53BP1 recruitment to DNA DSBs (Kolas et al. 2007; Wang and Elledge 2007) and additionally the residues of histone 4 lysine 20 and/or histone 3 lysine 79 have to be methylated (Huyen et al.2004;
Botuyan et al. 2006).
Notably, the phosphorylation of H2AX is not necessary for the initial 53BP1 recruitment instead this histone mark is implicated for 53BP1 retention to DSBs.
Furthermore Tip60 HAT activity is required for histones acetylation that might help 53BP1 recruitment.
In yeast, 53BP1 homologues Rad9 and Crb2 have indispensable roles in DNA-damage signaling and cell cycle arrest (Weinert and Hartwell 1988; Saka et al.
1997). Rad9 in particular is necessary for the G1, intra-S-phase and G2/M checkpoints. In mammalian cells, abrogation of 53BP1 function leads to defects in the G2 and intra-S-phase DNA damage checkpoints, but the magnitude of the defect is limited, and in chicken cells, in which the 53BP1 gene has been deleted, there is no G2 or intra-S-phase checkpoint defect (DiTullio et al. 2002; Wang et al. 2002;
Ward et al. 2003; Nakamura et al. 2006). This makes the role of 53BP1 in higher cells, difficult to interpret. It seems that 53BP1 may act redundantly with other proteins during DNA-damage signaling.
1.3.2 TopBP1 (Topoisomerase II β Binding Protein 1)
TopBP1 was first identified as a DNA topoisomerase II β binding protein
(Yamane et al. 1997). Human TopBP1 encodes eight BRCT (BRCA1 Breast Cancer susceptibility gene 1) domains. These domains were first discovered at the C- terminus of BRCA1 protein and they are commonly found in proteins that are implicated in the DNA damage response (Callebaut and Mornon 1997).
TopBP1 shares sequence homology with Saccharomyces cerevisiae Dpb11, Schizosaccharomyces pombe Rad4/Cut5, Drosophila Melanogaster Mus101 and Xenopus Cut5 and all these homologs are required for cell survival, DNA replication and DNA damage response (Makiniemi et al. 2001; Yamane et al. 2002; Garcia et al.
2005). When compared to vertebrate TopBP1 (eight BRCT domains), lower metazoan and yeast counterparts, are relatively smaller (respectively 7 and 4 BRCT domains). It appears that the higher eukaryotes have gained additional BRCT repeats to allow more complexity (Garcia et al. 2005).
Identification of TopBP1 as protein implicated in the DNA Damage Response. It has been shown that BRCA1 and TopBP1 colocalize after hydroxyurea treatment suggesting that TopBP1 plays a role at site of stalled replication forks (Makiniemi et al. 2001; Yamane et al. 2002). Furthermore induction of DNA DSBs also leads to TopBP1 focus formation (Makiniemi et al. 2001) and this reinforced the hypothesis that TopBP1 might be implicated in the DDR. More evidence for these notions were provided by showing that TopBP1 interacts with the phosphorylated C-terminus of
Rad9 (Makiniemi et al. 2001; Greer et al. 2003; Delacroix et al. 2007). Finally the finding that human TopBP1 activates the ATR/ATRIP complex (Kumagai et al.
2006) during replication stress, was an important discovery for recent studies.
Activation of ATR/ATRIP complex by TopBP1. TopBP1 colocalize with RPA and the ATR/ATRIP complex at sites of DNA replication stress (Makiniemi et al. 2001;
Garcia et al. 2005). Recently it has been found that TopBP1 works as a powerful promoter for ATR activation. The ATR Activating Domain (AAD) has been mapped, between the BRCT domains 6and 7 (Kumagai et al. 2006). Downregulation of TopBP1 prevents phosphorylation of ATR kinase targets like Chk1, Nbs1 and H2AX (Liu et al. 2006a). The mechanism of ATR activation by TopBP1 is still poorly understood, however it is probably due to a conformational change of the ATR/ATRIP complex promoted upon TopBP1 binding. It has been demonstrated that once TopBP1 is separated from the ATR/ATRIP complex, there is a reversion of the kinase activity to lower levels (Kumagai et al. 2006).
Further studies provided a description of ATR/ATRIP binding to TopBP1. In particular TopBP1 interacts directly with a region of ATRIP at its N-terminus, and this association facilitates the interaction between TopBP1 and ATR, enhancing its activation (wanMordes et al. 2008). Mutation in the TopBP1 interaction region of ATRIP impaired the ability to recover from replication stress and to induce cell cycle arrest.
The correct association between ATR and TopBP1 is under control of the ring complex Rad9-Hus1-Rad1 (Delacroix et al. 2007). Rad9 phosphorylated on Ser 387
binds TopBP1 BRCT domains 1 and 2. This interaction directs TopBP1 to the vicinity of ATR/ATRIP (Delacroix et al. 2007; Lee et al. 2007; Yan and Michael 2009) at the sites of stalled replication forks.
In both Xenopus and humans, TopBP1 BRCT domains 1 and 2 also interact with Nbs1 at the site of DNA DSBs (Morishima et al. 2007; Yoo et al. 2009). This interaction facilitates the recruitment of TopBP1 to sites of DNA DSBs. Further it has been proposed that once TopBP1 is recruited to sites to DNA DSBs, it is phosphorylated by ATM at Ser 1138 and this strongly enhances ATR activation.
However another study showed that TopBP1 focus formation depends only on BRCT 5 (Yamane et al. 2002).
Thus it becomes important to understand this discrepancy and why different groups have reported different results (discussed in Chapter 3).
Role of TopBP1 in DNA replication. During the G1 phase, ORC1, Cdt1, Cdc6 and the MCM proteins are sequentially assembled on replication origins to form the pre- replicative complex (pre-RC). The pre-RC is converted to an initiation complex (IC) at the beginning of S-phase by Cyclin Dependent Kinases (CDK) and the Cdc7-Dbf4 kinase (DDK). This leads to the recruitment of Cdc45 and then of Pol α and Pol ε at the replication origin (Diffley 2004).
In yeast, Dpb11 binds the replication initiation factors Sld2 and Sld3 (Synthetically Lethal with Dpb11), when they are phosphorylated by CDK at the beginning of S- phase (Tanaka et al. 2007a; Zegerman and Diffley 2007). This complex controls the
association with Cdc45 and the DNA polymerases at the replication origin (Masumoto et al. 2000). The phosphorylation of Sld proteins and the binding with Dpb11 represent the minimal requirement for the CDK-dependent activation of DNA replication initiation (Tanaka et al. 2007a; Tanaka et al. 2007b).
A similar role emerges when considering metazoan homologs of TopBP1. Cut5 in Xenopus, appears to be essential for S-phase CDK-dependent initiation of DNA replication and its depletion prevents binding between Cdc45 and DNA polymerases (Van Hatten et al. 2002; Hashimoto and Takisawa 2003). It has been shown also that Cut5 interacts with the replication protein RecqL4, a possible homolog candidate of Sld2, showing again similarities between metazoans and yeast (Sangrithi et al. 2005;
Matsuno et al. 2006).
A role of TopBP1 in initiation of DNA replication in mammals also has been demonstrated (Makiniemi et al. 2001). The VI BRCT domain seems particularly important for the replication function, as it is involved in the loading of Cdc45 to replication complexes (Schmidt et al. 2008). Moreover there are other proteins, recently discovered, that interact with TopBP1, like GEMC1 (Geminin coiled coil containing protein 1). GEMC1 resembles Sld3 because it interacts with Cdc45 and TopBP1; it is involved in the recruitment of Cdc45 onto replication origins and its phosphorylation by CDK, increases its affinity for TopBP1 and stimulates Cdc45 recruitment (Balestrini et al.; Piergiovanni and Costanzo 2010). A second interesting protein is represented by TRESLIN (Topbp1 interacting protein) that binds the BRCT domains 1 and 2 of TopBP1, and it acts in collaboration with it for the
loading of Cdc45 onto chromatin (Kumagai et al. 2010). The interaction between TopBP1 and TRESLIN is mediated by CDK dependent phosphorylation of TRESLIN (Kumagai et al. 2010). TRESLIN has been considered as another potential functional homolog of Sld3, in particular because TRESLIN unlike GEMC1, has sequence homology with Sld3. Yet the level of the homology is low, suggesting that particular replication factors such as Sld2 or Sld3 may have been subject to a rapid divergent evolution.
1.3.3 MDC1 (Mediator of DNA Damage Checkpoint 1)
Like 53BP1, MDC1 localizes rapidly to sites of DSBs following ionizing radiation (Shang et al. 2003). MDC1 foci colocalize with several other proteins implicated in DDR such 53BP1, γH2AX, and the MRN complex (Goldberg et al.
2003; Shang et al. 2003).
MDC1 is composed of different domains which are implicated in specific protein- protein interactions during the DNA-damage signaling.
Like 53BP1, MDC1 possesses two tandem BRCT domains located at the very C- terminus of the protein, between amino acid 1881 and 2082. In addition, MDC1 possesses a phospho-binding forkhead-associated (FHA) domain situated between amino acid 55-124, found already in other DNA damage proteins, such Nbs1 and Chk2, and finally MDC1 also has a proline-serine-threonine-rich PST domain and S/TQ clusters at its N-terminus (Stewart et al. 2003).
Unlike 53BP1, the BRCT domains of MDC1 are required for its recruitment to DNA DSBs (Goldberg et al. 2003; Shang et al. 2003) and this was later shown to occur via interaction with phopshorylated Ser 139 of H2AX. Point mutations in the BRCT domains of MDC1 as well as in the C-terminal part of H2AX abrogated the MDC1- γH2AX interaction (Stucki and Jackson 2006). This finding strongly indicates that
MDC1 could be the first protein to recognize the γH2AX chromatin mark, for the consequential recruitment and retention of the other DDR factors.
Following DNA DSB induction, the MRN complex recognizes the break and facilitates the localized activation of the kinase ATM, which rapidly phosphorylates H2AX (Uziel et al. 2003). This allows recruitment of MDC1 at the DNA damage sites. Once recruited, MDC1 functions as a molecular scaffold to stabilize the MRN complex bound to the DNA breaks, promoting the further accumulation of MRN (Stucki and Jackson 2006).
In addition, recent studies of human MDC1 have revealed that S/TQ motifs are the ATM target in response to IR (Kolas et al. 2007; Mailand et al. 2007; Matsuoka et al. 2007).
Phosphorylation of these sites mediates the recruitment of the E3 ubiquitin ligase RNF8 through direct interaction of its FHA domain with phosphorylated MDC1 (Huen et al. 2007; Kolas et al. 2007; Matsuoka et al. 2007). Immunofluorescence microscopy showed that deletion of these S/TQ motifs in MDC1 or depletion of RNF8 impaired the foci formation for conjugated ubiquitin, 53BP1 and BRCA1.
1.3.4 Claspin
Claspin was originally isolated as a protein that associates with recombinant GST-Chk1 in Xenopus (Kumagai and Dunphy 2000). Claspin possesses, like many proteins implicated in DDR, a large number of SQ/TQ motifs, which are potential substrates for kinases such as ATM and ATR (Kastan et al. 2000; Chini and Chen 2003).
Binding between Claspin and Chk1 occurs in response to DNA damage and is dependent on Claspin phosphorylation (Chini and Chen 2003) and this interaction is required for Chk1 activation and checkpoint control.
Experiments with Claspin siRNA demonstrated that human Claspin is an important key regulator of the DNA damage/replication checkpoint responses controlled by Chk1. One role of claspin is the inhibition of DNA synthesis induced by DNA damage. Another funtion mediated by Claspin and Chk1 is prevention of premature mitosis and finally Claspin participates even in the S-phase checkpoint.
Still the precise function of Claspin at the molecular level remains elusive.
1.3.5 RNF8 (RING Finger 8)
RNF8 is an E3 ubiquitin ligase that catalyzes regulatory ubiquitylation at the DNA lesions. RNF8 ubiquitin ligase activity mediates the accumulation of 53BP1 at DNA DSBs (Huen et al. 2007; Sakasai and Tibbetts 2008).
RNF8 possesses an FHA domain at its N-terminus and a RING finger motif at its C- terminus. This domain architecture is shared with the mitotic checkpoint regulator CHFR (Checkpoint with Fork-head associated and Ring Finger) (Scolnick and Halazonetis 2000).
RNF8 rapidly accumulates into IR-induced foci that colocalize with γH2AX (Huen et al. 2007; Wang and Elledge 2007).
The ability of RNF8 to form foci depends on interaction of its FHA domain with TQXF motifs at the N-terminus of MDC1 which are phosphorylated by ATM. (Huen et al. 2007; Mailand et al. 2007). RNF8 once recruited to site DNA DSBs, ubiquitylates both H2A and H2AX.
RNF8 physically interacts with UBC13 an E2 ubiquitin-conjugating enzyme (Plans et al. 2006).
RNF8 collaborates also with RNF168, a RING-type ubiquitin ligase protein (Doil et al. 2009; Stewart et al. 2009). Depletion of RNF168 displays a phenotype that is almost identical to that of RNF8, called RIDDLE syndrome, in which the cells are defective in DNA damage-induced regulatory ubiquitylation.
RNF168 accumulates at the sites of DNA damage within minutes following the induction of DNA DSBs (Doil et al. 2009; Stewart et al. 2009) and this recruitment is MDC1 and γH2AX dependent. Thus RNF8 accumulates first at the DNA DSBs, followed by RNF168 and BRCA1 (Doil et al. 2009). It is thought that ubiquitylation of H2A and H2AX by RNF8 and RNF168 affect chromatin structure in order to facilitate or allow 53BP1 interaction with methylated histones at the DNA damage sites (Stewart et al.2009).
1.4 Transducers
Transducers include protein kinases that, when activated by the presence of DNA damage, initiate a signal transduction cascade that propagates and amplifies the damage signal. Examples of transducers are the Chk1 and Chk2 kinases.
1.4.1 Chk1 and Chk2
In humans, there are two kinases. Chk1 and Chk2, with transduction functions in cell cycle regulation and checkpoint responses. (Walworth et al. 1993;
Rhind and Russell 1998; Melo and Toczyski 2002).
Chk1 is one of the most important substrates of ATR and its phosphorylation is mediated by the interaction with Claspin (Kumagai and Dunphy 2000; Kumagai and Dunphy 2003; Liu et al. 2006b). ATR phosphorylates Chk1 on Ser 317 and Ser 345 (Zhao and Piwnica-Worms 2001).
Both the ATM/Chk2 and ATR/Chk1 pathways converge to inactivate members of the CDC25 family of phosphatases, which play an important role in driving the cell- cycle transition (Donzelli and Draetta 2003).
Chk2 is activated by ATM in response to DNA DBS. It is phosphorylated on Thr 68 (Melchionna et al. 2000; Schwarz et al. 2003) which triggers a chain of
autophosphorylarion on Thr 383 and Thr 387 in the activation loop, ultimately resulting in Chk2 activation (Lee and Chung 2001; Schwarz et al. 2003).
1.5 Effectors
The effectors proteins groups represent the most downstream targets of the transducer protein kinases. The effectors typically affect cell cycle progression or promote DNA repair (Abraham 2001; Nyberg et al. 2002; Sancar et al. 2004).
Members of this group are the transcription factor p53 and the family of CDC25 phosphatases.
1.5.1 CDC25 family
The two phosphatases CDC25A and CDC25C are phosphorylated by Chk1 and Chk2 after DNA damage. CDC25A is marked for degradation upon phosphorylation by Ch1k on Ser 123 (Falck et al. 2001; Zhao et al. 2002).
Otherwise, CDC25A dephosphorylates the cyclin-dependent kinases CDK1 and CDK2, thereby maintaining them in their activated states, which allow progression of the cell cycle through the G2/M and G1/S transitions, respectively (Mailand et al.
2000; Mailand et al. 2002). CDC25C is phosphorylated by Chk1 and Chk2 on Ser
216, thereby creating a binding site for 14-3-3 protein, which subsequently inhibits it or targets it for degradation (Peng et al. 1997; Brown et al. 1999).
1.5.2 p53
p53 is called the “guardian of the genome” (Lane 1992). It can be activated in response to DNA damage, oncogene activation or hypoxia and it orchestrates biological outputs like cell-cycle arrest, apoptosis, senescence or modulation of autophagy (Yee and Vousden 2005; Riley et al. 2008; Green and Kroemer 2009).
p53 is negatively regulated through its interaction with Mdm2, which mediates ubiquitin-mediated degradation of p53.
In response to DNA damage from ionizing radiation or other agents, p53 is phosphorylated at its N-terminal by several kinases like ATM, ATR, DNA-PK, Chk1 and Chk2 (Appella and Anderson 2001). The phosphorylation of p53 prevents Mdm2 binding, resulting in the stabilization of p53. Following its stabilization, p53 can freely bind DNA in a sequence-specific manner (el-Deiry et al. 1992) and activates or represses its target genes. One of the target genes is p21, which inhibits the CDK2-cyclin E complex, arresting the cells at the G1/S border (Banin et al.
1998).
1.6 The G1 checkpoint
Most eukaryotic cells damaged in G1, exhibits a pronounced delay prior to entry into the S-phase. This arrest in G1 allows vital time for repair and prevents replication of damaged DNA.
The most important event in the G1-S checkpoint is the stabilization of p53, which in turn induces transcription of p21, an inhibitor of the cyclin E-CDK2 complex.
ATM is implicated in p53 regulation by phosphorylating it on its Ser 15, which destabilizes the binding of p53 to Mdm2. Additionally, the mechanism for destabilization of p53, involves Chk2, which relays ATM-dependent signals to p53 and many other downstream targets in IR-damaged cells (Ahn et al. 2000;
Melchionna et al. 2000). Chk2 phosphorylates p53 on Ser20 which directly interferes with the binding of p53 to Mdm2 (Figure 2).
These findings demonstrate that ATM establishes multiple regulatory contacts with p53 for the activation of G1 checkpoint. In contrast, the potential role of the ATR/Chk1 pathway for the G1 checkpoint remains obscure. It is conceivable that a parallel pathway, mediated by ATR, for the p53 activation in G1 might also exist.
1.7 The intra-S-checkpoint
The intra-S-checkpoint is activated by DNA damage encountered during DNA (Paulovich and Hartwell 1995).
Exposure to IR, during S phase, activates ATM dependent pathway leading to the degradation of Cdc25A and a consequential inhibition of DNA synthesis (Donaldson and Blow 1999; Takisawa et al. 2000).
When DNA is damaged by UV light or chemicals during S phase then the ATR/ATRIP complex is activated (Abraham 2001; Cortez et al. 2001). The activation of the ATR-dependent pathway relies on RPA, the 9-1-1- complex and TopBP1 described above and leads to downregulation of Cdc25A (Heffernan et al.
2002). Activation of the intra-S-checkpoint leads to a stabilization of DNA replication forks and inhibition of firing of late origins (Figure 2).
1.8 The G2 checkpoint
The G2/M checkpoint prevents cells from continuing cell cycle progression into mitosis in the presence of DNA damage.
ATR and Chk1 are the main players of this checkpoint, although ATM also contributes, particularly in the early response to DNA damage. Activation of Chk1 in G2 leads to phosphorylation of the mitosis-promoting phosphatase Cdc25C. Upon phosphorylation, the Cdc25C phosphatase binds the 14-3-3 proteins; it becomes sequestered in the cytoplasm, where it is eventually degraded (Furnari et al. 1997;
Peng et al. 1997; Sanchez et al. 1997). Inactivation of Cdc25 prevents entry into mitosis by keeping the mitotic cyclin B-Cdc2 inactive.
Although Chk2 could actually contribute to G2 checkpoint, ATR/Chk1 pathway remains the most important one (Figure 2).
Figure 1: Cellular responses to DNA damage: different types of DNA damage cause different types of lesions and these are handled in turn differently by the cells. The outcome is cell survival, cell death or malignant transformation (Shiloh 2003).
Figure 2: An overview of mammalian signal transduction pathways activating cell cycle checkpoint arrest after DNA damage: signal transduction pathways are activated by ATM and ATR. The signaling pathways involve the mediator proteins which amplify the signal, transducer kinases and effector proteins.
ATM activation by DSBs in G1 leads to Chk2 phosphorylation and subsequent phosphorylation of CDC25A. A second mechanism includes the tumor suppressor p53, which is activated directly or indireclty by ATM, and serves as a transcription factor of p21 which inhibit cell cycle progression.
Collapsed or stalled replication forks in S-phase activate ATR. This leads to Chk1 phosphorylation and subsequent phosphorylation and proteolysis of CDC25A. This prevents initiation of new replication origins.
In G2/M checkpoint there is activatin of both ATM and ATR. Similar to G1 checkpoint or S-checkpoint, the DNA damage response is initiated by the phosphorylation of checkpoint kinases Chk1 and Chk2 and phosphatases (CDC25). This prevents dephosphorylation of CDK1-cyclin B, which is needed for entry in mitosis (Lobrich and Jeggo 2007).
CHAPTER 2
MATERIALS AND METHODS
2.1 Cell lines
Cell lines were obtained from the following sources: U2OS cells from American Type Culture Collection (Manassas VA, USA); H2AX -/-, H2AX +/+ and MDC1 -/- mouse embryonic fibroblasts (MEFs) were a gift from Manuel Stucki (Universty of Zurich, Switzerland).
2.2 Recombinant plasmids and antibodies
Plasmids encoding TopBP1 polypeptides fused to the C-terminus of Green Fluorescent Protein (GFP) were generated from a previously-described mammalian expression plasmid (Huyen et al. 2004). For stable expression the GFP-TopBP1 inserts were transferred from the pSNV2 vector to the pIRESN2 bicistronic vector (Clontech Laboratories, Mountain View, CA) (Protocol N°7).
For the co-immunoprecipitation experiments, the pIRESN2 vector was used to express a fusion protein consisting from its N-terminus to C-terminus of an HA tag, two tandem IgG-binding domains from protein A (Nilsson et al, 1987), a heterologous tetramerization (TZp) fused to a nuclear localization signal (Zgheib et
al. 2009) and residues 2-300 or 531-755 of human TopBP1. Antibodies used were specific for 53BP1 (Schultz et al. 2000), ATRIP (Venere et al. 2007), GFP, ATM and TopBP1 (Abcam, Cambridge, United Kingdom), RPA (Calbiochem, La Jolla, CA, USA and Genetech, South San Francisco, CA, USA), Cyclin B1 (Upstate, Albany, NY, USA) and the HA tag (Covance, Princeton, NJ, USA).
2.3 Focus formation assay and immunofluoresce
For immunofluoresence experiments (protocol N°1), U2OS grown on coverslips were either not transfected (to monitor localization of endogenous proteins) or were transiently transfected with plasmids encoding GFP-TopBP1 proteins using Fugene (Roche Diagnostic, Basel Switzerland) and examined two days later. To induce DNA damage, the cells were exposed to IR (9Gy), and, 30 minutes to several hours later, fixed and processed for immunofluorescence, as described (Zgheib et al. 2009). Alternatively, cells were treated with 2mM hydroxyurea (HU) and examined 16 hours later.
To correlate the behavior of TopBP1 foci according to genomic DNA content, overlapping images capturing a total of 227 cells were acquired using 40x magnification lens. The genomic DNA content of each cell was determined by intergrating the density of DAPI staining over the entire nucleus of the cell after the images had been calibrated (via their overlapping) to another. Then an istogram plot showing for each cell the genomic DNA content and the behavior of TopBP1 was
prepared. For all the immunofluorescence assays, the DNA content of the indicated cell was determined to be 2N or >2N based on the integrated DAPI staining value of the cell in comparison to the adjacent cells in the same field. For many experiments, the cells were also treated with EdU for 30 minutes prior fixing and the presence or absence of EdU incorporation, helped distinguish S phase cells from G1 or G2. The processing of the images and the calculations were performed using Imagevision/IRIX and PERL software. (Silicon Graphics Inc., Mountain View, CA, USA). For the correlations of Cyclin B1 staining and EdU incorporation to behavior of TopBP1 IR-induced foci, more than 200 cells were counted.
2.4 Small interfering RNA (siRNA) Transfection
U2OS were transfected using Oligofectamine (Invitrogen, Carlsbad, CA, USA) or Hyperfectamine (Qiagen, Valencia, CA, USA) (Protocol N°2) with control siRNA (luciferase, Dharmacon, Lafayette, Co, USA) or siRNA targeting atm, 53BP1, mdc1 (Mochan et al. 2003), atrip (Venere et al. 2007), rpa70 or TopBP1.
The sequence targeting rpa70 was: ACCACTCTATCCTCTTTCATGdtdt. For TopB1 three differents siRNA were used: #1, CAGAAUUGUUGGUCCUCAAdtdt,
#2, GAACCCUGCUUCAGAGUAUAdtdt and #3,
CGAUAGAGGAGACUCAUGAdtdt. The cells were examined 48 or 72 hours after siRNA transfection.
2.5 Preparation of the cell extracts, immunoblotting and coprecipitation assays
The preparation of the whole cell extracts and immunoblotting were performed as described in Protocol N°3 (Venere et al. 2007). For analysis of the interaction of the BRCT domains of TopBP1 with endogenous 53BP1, stable clones of U2OS cells expressing the pIRESN2 vectors described above were selected with G418 as described (Venere et al. 2007). Nuclear extracts prepared from these cells (Dignam 1983) were incubated for 1 hour at 4°C in buffer consisting of 25mM BTP (pH 6.8), 10mM EDTA, 1mM EGTA, 150mM KCl, 5% Glycerol and 0.75%
CHAPS with M-270 Epoxy Dynabeads (Dynal AS; Oslo, Norway) coated with rabbit IgG (Sigma, St Louis, MO, USA). Then the beads were washed extensively in the same buffer and captured HA-tagged TopBP1 and endogenous 53BP1 proteins were detected by immunoblotting (Protocols N°4 and N°5).
2.6 Checkpoint assay and FACS analysis
48 hours after siRNA transfection, U2OS cells pulse-labeled for 1 hour with 10µM EdU (Invitrogen) were exposed to IR (2Gy) or were mock irradiated. Then
the cells were incubated for 7 hours with 10µM BrdU (Sigma) and 0.25µg/ml Nocodazole (Sigma).
Afterwards, the cells were harvested by trypzinization and fixed in ice cold 70%
ethanol for 20 minutes. The presence of EdU and BrdU were monitored by Click-It
chemistry (Invitrogen) and anti-BrdU antibodies (BD Bioscences, Franklin Lakes, NJ, USA) respectively. After staining of the genomic DNA with propidium iodide, the samples were analyzed by flow cytometry (Protocol N°6).
Protocol N°1: Immufluorescence
Transfer coverslips to multiwell plate (DMEM)
Irradiate if nceserrary and reincubate at 37°C
Remove the medium and wash with 0.5ml PBS 1x
Remove PBS and fix the cells with ice cold methanol (-20°C). Keep at -20°C for 10min.
Remove methanol and rinse twice with PBS 1x
Add 0.5ml permeabilization solution buffer (0.2% -Triton X-100 in PBS 1x).
Incubate 10min at 4°C.
Wash twice with 1ml PBS 1x and continue with EdU or normal Ab.
For EdU: add 200µl EdU cocktail Invitrogen kit
Number of samples 5 10 15 20 25 30
1x Click-iT rxn bu (100 mM Tris pH 8.0)
860µl 1720µl 2580µl 3440µl 4300µl 5160µl
CuSO4 (100 mM CuSO4) 40µl 80µl 120µl 160µl 200µl 240µl Alexa Fluor 647
(0.5mg in 70 l DMSO)
2.5µl 5µl 7.5µl 10µl 12.5µl 15µl
Additif (1M Na Ascorbate in water)
100µl 200µl 300µl 400µl 500µl 600µl
Total volume 1ml 2ml 3ml 4ml 5ml 6ml
Incubate 30min in the dark at Room Temperature (R.T)
Block with PBS 1x BSA 1% for 20min at R.T.
Add 0.5ml Primary Antibody per well diluted in PBS 1x BSA 1%. Incubate 1h at R.T or over night at 4°C.
Wash twice with PBS 1x BSA 1%
Add Secondary Antibody diluted in PBS 1x BSA 1% for 30min in the dark at R.T.
Wash twice with PBS 1x
Add DAPI (1µg/ml) in the dark at R.T for 1min
Rinse twice with PBS 1x
Take coverslips with tweezers and rinse in 12ml falcon tube containing water.
Touch side of coverslips with Kimwipe to remove excess of water.
Mount coverslips upside down on slide, on top of a drop of Fluoromont.
Let dry in a drawer for a few hours or better over night.
Protocol N° 2: DNA transfection or siRNA Transfection
For 6 well plates:
Per well:
Mix in Eppendorf: per wells (6l Fugene + 50l OPTIMEM).
Add 50l OPTIMEM to 2 g DNA in other Eppendorf. (1g each in case of multiple transfections).
Leave 15min at R.T.
Add 50l FuGene-OPTIMEM to each DNA tube
Leave 15min at R.T.
Remove media from 6well plate.
Add to each well 2ml of OPTIMEM + 2% FBS.
Add DNA/FuGene/OPTIMEM to wells.
Incubate overnight 37°C.
Replace next day media with DMEM.
siRNA Oligofectamine protocol (Invitrogen, Carlsbad, CA, USA)
Day 0: for U2OS cells split 120.000 cells per 60mm dish.
Day1: per 60mm dish
Tube 1: 12µl 20µM siRNA + 200µl room temperature OPTIMEM Tube 2: 12µl Oligofectamine + 48µl room temperature OPTIMEM
Incubate Tube2 for 7min in the tissue culture hood.
Add Tube 2 to Tube 1.
Incubate for 20min.
In the meantime change media in 60mm dish to 2.6ml OPTIMEM.
Add 128µl OPTIMEM to the mixture to 60mm dish. Wait 4 hours.
Add 1ml 40% FBS to the 60mm dish.
Day 4: the cells are ready for further experiments (W.B. or IF).
siRNA Hiperfectamine protocol (Qiagen, Valencia, CA, USA)
Split 2-4x106 U2OS cells in per 10cm dish in 7ml of DMEM.
For short time until transfection, incubate the cells under normal growth conditions
Diluite 600ng in 1ml culture medium without serum. Add 40µl of Hiperfectamine Transfection Reagent to the diluted siRNA and mix vortexing.
Incubate the samples 5-10min at R.T. to allow the formation of the transfection complexes.
Add the complex drop-wise onto cells. Gently swirl the dish to ensure uniform distribution of the transfection complexes.
Incubate the cells with the transfection complexes under their normal growth conditions and monitor gene silencing after an appropriate time.
Protocol N°3: Whole cell extract
Trypsinize the cells and transfer to the 15ml tube.
Spin down at 4°C 10min at 1200rpm.
Resuspend in ice cold PBS 1x and spin down again.
Remove PBS and resuspend in 1x EBC buffer (300µl/10cm dish) and
transfer to the eppendorf tube.
Incubate 45min at 4°C with rocking.
Add 1µl DNAase (RNAase free) and 1µl 0.5 Mg/Cl2 /100µl extract and
incubate 1h in 16°C waterbath.
Spin 15min at 15000rpm at 4°C, to remove cellular debris.
Aliquot supernatant in eppendorf and freeze or proceed with SDS-Page.
2xEBC buffer (100ml):
100 mM Tris pH 8.0 240mM NaCl
1% NP-40 or Tween 20 Water
Inhibitors to add to 1x EBC per ml 1µl 1M DTT
Add protease and phospatase inhibitors 4µl 100µM Wortmannin
40µl 100µM Caffein
Protocol N°4: Nuclear Extraction
Trypsinize the cells, spin down at 1000rpm at 4°C for 5min and resuspend cell in 40ml PBS 1x.
Spin cells and resuspend in 1ml Buffer A per 15cm plate. Transfer to 15ml tube.
Hold for 10min on ice. Add inhibitors to Buffer A at this time.
Spin cells for 10min. Resuspend in Buffer A to a equal ½ volume of dounce homogenizer (6ml). Transfer to homogenizer and lyse cells by 25 strokes with pestle B.
Transfer to 15ml tube and spin down at 3000rm at 4°C for 15min.
Depending on the number of plates:
If less than 4 plates resuspend pellet in 1ml Buffer A per plate, transfer to ultra-eppendorfs, spin for 10min at 40000rpm in TLA ultra.
If more than 4 plates resuspend pellet in 0.5ml Buffer A per plate, transfer to bottles for ultra, spin for 10min at 30000rpm in 50Ti rotor in big ultra.
Add inhinitors to Buffer B.
Resuspend pellter in 0.5 to 1 ml Buffer B and transfer to ultra-eppendorf.
Add DNAase 1µl for 100µl extract; incubate 1h at 16°C with shaking.
Spin for 10min at 40000rpm in TLA.
Split supernatant into tubes and freeze at -70°C.
Buffer A (25ml) Buffer B (10ml)
2µl 0.1M Zinc Acetate 0.8µl 0.1 Zinc Acetate
250µl 1M HEPES pH 7.9 200µl HEPES pH 7.9
75µl 0.5M MgCl2 30µl 0.5 MgCl2
250µl 1M KCl 3ml KCl
0.5ml 0.5 NaPO4 pH 8 0.2ml 0.5M NaPO4
24.5ml H2O 4.1ml H2O
Inhibitors per ml buffers (Add just before to use) 1µl 1M DTT
Proteases and phosphatases inhibitors 4µl Wortmannin
Protocol N°5: Preparation of IgG dynabeads
Coat 10mg epoxy-dynabeads (6.6x108; Dynal M-270 Epoxy 143.01). Needed 3µg ligand (rabbit IgG)/107 beads. Therefore needed 198µg ligand.
Prepare 1mg/ml stock of rabbit-IgG (Sigma I-5006 reagent grade) in PB1 1x (0.1M Na3PO4 buffer pH 5.4, 150mM NaCl).
Resuspend 10mg beads in 600µl Na3PO4 buffer. Wash 2more times in the same buffer.
Resuspend beads with 198µl Na3PO4 buffer
Add 198µl of ligand
Add 198µl 3M (NH4)2 SO4 (ammonium sulphate).
Incubate 24h with slow tilt rotationat 37°C.
Place tube in magnet for 4min, remove the supernatant.
Wash 4x 10min PBS 1x.
Resuspend beads in 1.2ml PBS +0.02% sodium azide
Store at 4°C for about 2 weeks.
Purification of protein G complexes with IgG-dynabeads
Prewashed IgG-dynabeads 2x IP-buffer1
Use about 50mg protein. Keep an amount for the INPUT.
Add 554µl Ig-G dynabeads (slurry).
Incubate on nutator for 1.5h at 4°C.
Remove supernatant, keep an amount of FLOWTHROUGH.
Wash 6x 1ml IP-buffer2 at 4°C.
Resuspend beads in 30µl SDS-PAGE loading buffer without β-ME.
Heat beads 20min at 65°C, remove the supernatant to fresh tube.
Add β-ME to the supernatant.
Boil supernatant 4min at 95°C.
Load on 12% SDS-PAGE along with INPUT and FLOWTHROUGH.
IP-buffer 1 (IP-buffer2 is the same but without the detergent and salt)
25mM BTP pH 6.8 10mM EDTA 1mM EGTA 150mM KCl 5% Glycerol 1% Triton X-100
Add proteases inhibitors.
