A hallmark of the human cell cycle in normal somatic cells is its precision. This remarkable fidelity is achieved by a number of signal transduction pathways, known as checkpoints, which monitor cell cycle progression ensuring an interdependency of S-phase and mitosis, the integrity of the genome and the fidelity of chromosome segregation.
Checkpoints are layers of control that act to delay CDK activation when defects in the division program occur. As the CDKs functioning at different points in the cell cycle are regulated by different means, the various checkpoints differ in the biochemical mechanisms by which they elicit their effect. However, all checkpoints share a common hierarchy of a sensor, signal transducers, and effectors that interact with the CDKs.<p>The stability of the genome in somatic cells contrasts to the almost universal genomic instability of tumor cells. There are a number of documented genetic lesions in checkpoint genes, or in cell cycle genes themselves, which result either directly in cancer or in a predisposition to certain cancer types. Indeed, restraint over cell cycle progression and failure to monitor genome integrity are likely prerequisites for the molecular evolution required for the development of a tumor. Perhaps most notable amongst these is the p53 tumor suppressor gene, which is mutated in >50% of human tumors. Thus, the importance of the checkpoint pathways to human biology is clear.
View original pathway at Reactome.</div>
Ball HL, Cortez D.; ''ATRIP oligomerization is required for ATR-dependent checkpoint signaling.''; PubMedEurope PMCScholia
Espinosa JM, Verdun RE, Emerson BM.; ''p53 functions through stress- and promoter-specific recruitment of transcription initiation components before and after DNA damage.''; PubMedEurope PMCScholia
Namiki Y, Zou L.; ''ATRIP associates with replication protein A-coated ssDNA through multiple interactions.''; PubMedEurope PMCScholia
Falck J, Mailand N, Syljuåsen RG, Bartek J, Lukas J.; ''The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis.''; PubMedEurope PMCScholia
Linares LK, Hengstermann A, Ciechanover A, Müller S, Scheffner M.; ''HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53.''; PubMedEurope PMCScholia
Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K, Elledge SJ.; ''Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro.''; PubMedEurope PMCScholia
Sironi L, Mapelli M, Knapp S, De Antoni A, Jeang KT, Musacchio A.; ''Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a 'safety belt' binding mechanism for the spindle checkpoint.''; PubMedEurope PMCScholia
Zou L, Liu D, Elledge SJ.; ''Replication protein A-mediated recruitment and activation of Rad17 complexes.''; PubMedEurope PMCScholia
Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ.; ''The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.''; PubMedEurope PMCScholia
Danielsen JR, Povlsen LK, Villumsen BH, Streicher W, Nilsson J, Wikström M, Bekker-Jensen S, Mailand N.; ''DNA damage-inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger.''; PubMedEurope PMCScholia
Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY, Taya Y, Prives C, Abraham RT.; ''A role for ATR in the DNA damage-induced phosphorylation of p53.''; PubMedEurope PMCScholia
Bermudez VP, Lindsey-Boltz LA, Cesare AJ, Maniwa Y, Griffith JD, Hurwitz J, Sancar A.; ''Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro.''; PubMedEurope PMCScholia
Ciccia A, Elledge SJ.; ''The DNA damage response: making it safe to play with knives.''; PubMedEurope PMCScholia
Unsal-Kaçmaz K, Sancar A.; ''Quaternary structure of ATR and effects of ATRIP and replication protein A on its DNA binding and kinase activities.''; PubMedEurope PMCScholia
Momand J, Zambetti GP, Olson DC, George D, Levine AJ.; ''The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation.''; PubMedEurope PMCScholia
Chen L, Gilkes DM, Pan Y, Lane WS, Chen J.; ''ATM and Chk2-dependent phosphorylation of MDMX contribute to p53 activation after DNA damage.''; PubMedEurope PMCScholia
Cotta-Ramusino C, McDonald ER, Hurov K, Sowa ME, Harper JW, Elledge SJ.; ''A DNA damage response screen identifies RHINO, a 9-1-1 and TopBP1 interacting protein required for ATR signaling.''; PubMedEurope PMCScholia
Clarke CA, Clarke PR.; ''DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system.''; PubMedEurope PMCScholia
Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, Smorodinsky NI, Prives C, Reiss Y, Shiloh Y, Ziv Y.; ''Enhanced phosphorylation of p53 by ATM in response to DNA damage.''; PubMedEurope PMCScholia
Monte M, Benetti R, Collavin L, Marchionni L, Del Sal G, Schneider C.; ''hGTSE-1 expression stimulates cytoplasmic localization of p53.''; PubMedEurope PMCScholia
Foray N, Marot D, Gabriel A, Randrianarison V, Carr AM, Perricaudet M, Ashworth A, Jeggo P.; ''A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein.''; PubMedEurope PMCScholia
Zhao H, Piwnica-Worms H.; ''ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1.''; PubMedEurope PMCScholia
Huang L, Yan Z, Liao X, Li Y, Yang J, Wang ZG, Zuo Y, Kawai H, Shadfan M, Ganapathy S, Yuan ZM.; ''The p53 inhibitors MDM2/MDMX complex is required for control of p53 activity in vivo.''; PubMedEurope PMCScholia
Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM.; ''Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53.''; PubMedEurope PMCScholia
Hang H, Lieberman HB.; ''Physical interactions among human checkpoint control proteins HUS1p, RAD1p, and RAD9p, and implications for the regulation of cell cycle progression.''; PubMedEurope PMCScholia
Geyer RK, Yu ZK, Maki CG.; ''The MDM2 RING-finger domain is required to promote p53 nuclear export.''; PubMedEurope PMCScholia
Wang B, Matsuoka S, Ballif BA, Zhang D, Smogorzewska A, Gygi SP, Elledge SJ.; ''Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response.''; PubMedEurope PMCScholia
Sar F, Lindsey-Boltz LA, Subramanian D, Croteau DL, Hutsell SQ, Griffith JD, Sancar A.; ''Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures.''; PubMedEurope PMCScholia
Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ.; ''Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.''; PubMedEurope PMCScholia
Li J, Stern DF.; ''DNA damage regulates Chk2 association with chromatin.''; PubMedEurope PMCScholia
Campbell MS, Chan GK, Yen TJ.; ''Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase.''; PubMedEurope PMCScholia
Chehab NH, Malikzay A, Appel M, Halazonetis TD.; ''Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53.''; PubMedEurope PMCScholia
Plafker SM, Plafker KS, Weissman AM, Macara IG.; ''Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.''; PubMedEurope PMCScholia
Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, Appella E, Kastan MB, Siliciano JD.; ''Activation of the ATM kinase by ionizing radiation and phosphorylation of p53.''; PubMedEurope PMCScholia
Bulavin DV, Higashimoto Y, Demidenko ZN, Meek S, Graves P, Phillips C, Zhao H, Moody SA, Appella E, Piwnica-Worms H, Fornace AJ.; ''Dual phosphorylation controls Cdc25 phosphatases and mitotic entry.''; PubMedEurope PMCScholia
Peng CY, Graves PR, Thoma RS, Wu Z, Shaw AS, Piwnica-Worms H.; ''Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216.''; PubMedEurope PMCScholia
Pant V, Xiong S, Iwakuma T, Quintás-Cardama A, Lozano G.; ''Heterodimerization of Mdm2 and Mdm4 is critical for regulating p53 activity during embryogenesis but dispensable for p53 and Mdm2 stability.''; PubMedEurope PMCScholia
Graves PR, Lovly CM, Uy GL, Piwnica-Worms H.; ''Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein binding.''; PubMedEurope PMCScholia
Chang LF, Zhang Z, Yang J, McLaughlin SH, Barford D.; ''Molecular architecture and mechanism of the anaphase-promoting complex.''; PubMedEurope PMCScholia
Jazayeri A, Falck J, Lukas C, Bartek J, Smith GC, Lukas J, Jackson SP.; ''ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks.''; PubMedEurope PMCScholia
Lakin ND, Hann BC, Jackson SP.; ''The ataxia-telangiectasia related protein ATR mediates DNA-dependent phosphorylation of p53.''; PubMedEurope PMCScholia
Griffith JD, Lindsey-Boltz LA, Sancar A.; ''Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy.''; PubMedEurope PMCScholia
Byun TS, Pacek M, Yee MC, Walter JC, Cimprich KA.; ''Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.''; PubMedEurope PMCScholia
Li M, Luo J, Brooks CL, Gu W.; ''Acetylation of p53 inhibits its ubiquitination by Mdm2.''; PubMedEurope PMCScholia
Wilson KA, Stern DF.; ''NFBD1/MDC1, 53BP1 and BRCA1 have both redundant and unique roles in the ATM pathway.''; PubMedEurope PMCScholia
Sudakin V, Chan GK, Yen TJ.; ''Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.''; PubMedEurope PMCScholia
Monte M, Benetti R, Buscemi G, Sandy P, Del Sal G, Schneider C.; ''The cell cycle-regulated protein human GTSE-1 controls DNA damage-induced apoptosis by affecting p53 function.''; PubMedEurope PMCScholia
Montagnoli A, Fiore F, Eytan E, Carrano AC, Draetta GF, Hershko A, Pagano M.; ''Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation.''; PubMedEurope PMCScholia
Dalal SN, Schweitzer CM, Gan J, DeCaprio JA.; ''Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site.''; PubMedEurope PMCScholia
Melchionna R, Chen XB, Blasina A, McGowan CH.; ''Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1.''; PubMedEurope PMCScholia
Luo X, Tang Z, Rizo J, Yu H.; ''The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20.''; PubMedEurope PMCScholia
Shieh SY, Ahn J, Tamai K, Taya Y, Prives C.; ''The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites.''; PubMedEurope PMCScholia
Bochkareva E, Belegu V, Korolev S, Bochkarev A.; ''Structure of the major single-stranded DNA-binding domain of replication protein A suggests a dynamic mechanism for DNA binding.''; PubMedEurope PMCScholia
Chehab NH, Malikzay A, Stavridi ES, Halazonetis TD.; ''Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage.''; PubMedEurope PMCScholia
Peters JM.; ''The anaphase-promoting complex: proteolysis in mitosis and beyond.''; PubMedEurope PMCScholia
Khanna KK, Keating KE, Kozlov S, Scott S, Gatei M, Hobson K, Taya Y, Gabrielli B, Chan D, Lees-Miller SP, Lavin MF.; ''ATM associates with and phosphorylates p53: mapping the region of interaction.''; PubMedEurope PMCScholia
Blackwell LJ, Borowiec JA.; ''Human replication protein A binds single-stranded DNA in two distinct complexes.''; PubMedEurope PMCScholia
Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida H, Liu D, Elledge SJ, Mak TW.; ''DNA damage-induced activation of p53 by the checkpoint kinase Chk2.''; PubMedEurope PMCScholia
el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B.; ''WAF1, a potential mediator of p53 tumor suppression.''; PubMedEurope PMCScholia
Sharp DA, Kratowicz SA, Sank MJ, George DL.; ''Stabilization of the MDM2 oncoprotein by interaction with the structurally related MDMX protein.''; PubMedEurope PMCScholia
Oliner JD, Kinzler KW, Meltzer PS, George DL, Vogelstein B.; ''Amplification of a gene encoding a p53-associated protein in human sarcomas.''; PubMedEurope PMCScholia
Raderschall E, Golub EI, Haaf T.; ''Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage.''; PubMedEurope PMCScholia
Hopkins KM, Wang X, Berlin A, Hang H, Thaker HM, Lieberman HB.; ''Expression of mammalian paralogues of HRAD9 and Mrad9 checkpoint control genes in normal and cancerous testicular tissue.''; PubMedEurope PMCScholia
Fuchs SY, Adler V, Buschmann T, Wu X, Ronai Z.; ''Mdm2 association with p53 targets its ubiquitination.''; PubMedEurope PMCScholia
Blasina A, de Weyer IV, Laus MC, Luyten WH, Parker AE, McGowan CH.; ''A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase.''; PubMedEurope PMCScholia
Lieberman HB, Hopkins KM, Nass M, Demetrick D, Davey S.; ''A human homolog of the Schizosaccharomyces pombe rad9+ checkpoint control gene.''; PubMedEurope PMCScholia
Boyd SD, Tsai KY, Jacks T.; ''An intact HDM2 RING-finger domain is required for nuclear exclusion of p53.''; PubMedEurope PMCScholia
Parker LL, Piwnica-Worms H.; ''Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase.''; PubMedEurope PMCScholia
Fang G, Yu H, Kirschner MW.; ''The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation.''; PubMedEurope PMCScholia
Lee CH, Chung JH.; ''The hCds1 (Chk2)-FHA domain is essential for a chain of phosphorylation events on hCds1 that is induced by ionizing radiation.''; PubMedEurope PMCScholia
Chen J, Marechal V, Levine AJ.; ''Mapping of the p53 and mdm-2 interaction domains.''; PubMedEurope PMCScholia
Wei SJ, Williams JG, Dang H, Darden TA, Betz BL, Humble MM, Chang FM, Trempus CS, Johnson K, Cannon RE, Tennant RW.; ''Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation.''; PubMedEurope PMCScholia
Zou L, Elledge SJ.; ''Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.''; PubMedEurope PMCScholia
Cheng Q, Chen L, Li Z, Lane WS, Chen J.; ''ATM activates p53 by regulating MDM2 oligomerization and E3 processivity.''; PubMedEurope PMCScholia
McGowan CH, Russell P.; ''Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15.''; PubMedEurope PMCScholia
Scoumanne A, Cho SJ, Zhang J, Chen X.; ''The cyclin-dependent kinase inhibitor p21 is regulated by RNA-binding protein PCBP4 via mRNA stability.''; PubMedEurope PMCScholia
Galaktionov K, Beach D.; ''Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins.''; PubMedEurope PMCScholia
Cai Z, Chehab NH, Pavletich NP.; ''Structure and activation mechanism of the CHK2 DNA damage checkpoint kinase.''; PubMedEurope PMCScholia
Pereg Y, Shkedy D, de Graaf P, Meulmeester E, Edelson-Averbukh M, Salek M, Biton S, Teunisse AF, Lehmann WD, Jochemsen AG, Shiloh Y.; ''Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage.''; PubMedEurope PMCScholia
Sørensen CS, Syljuåsen RG, Lukas J, Bartek J.; ''ATR, Claspin and the Rad9-Rad1-Hus1 complex regulate Chk1 and Cdc25A in the absence of DNA damage.''; PubMedEurope PMCScholia
Maki CG.; ''Oligomerization is required for p53 to be efficiently ubiquitinated by MDM2.''; PubMedEurope PMCScholia
Cheng Q, Cross B, Li B, Chen L, Li Z, Chen J.; ''Regulation of MDM2 E3 ligase activity by phosphorylation after DNA damage.''; PubMedEurope PMCScholia
Ball HL, Myers JS, Cortez D.; ''ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation.''; PubMedEurope PMCScholia
Ellison V, Stillman B.; ''Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA.''; PubMedEurope PMCScholia
Iftode C, Daniely Y, Borowiec JA.; ''Replication protein A (RPA): the eukaryotic SSB.''; PubMedEurope PMCScholia
Wang W, Nacusi L, Sheaff RJ, Liu X.; ''Ubiquitination of p21Cip1/WAF1 by SCFSkp2: substrate requirement and ubiquitination site selection.''; PubMedEurope PMCScholia
Cordeiro-Stone M, Makhov AM, Zaritskaya LS, Griffith JD.; ''Analysis of DNA replication forks encountering a pyrimidine dimer in the template to the leading strand.''; PubMedEurope PMCScholia
Wang B, Matsuoka S, Carpenter PB, Elledge SJ.; ''53BP1, a mediator of the DNA damage checkpoint.''; PubMedEurope PMCScholia
Zhao H, Watkins JL, Piwnica-Worms H.; ''Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints.''; PubMedEurope PMCScholia
Voges D, Zwickl P, Baumeister W.; ''The 26S proteasome: a molecular machine designed for controlled proteolysis.''; PubMedEurope PMCScholia
Bernardi R, Liebermann DA, Hoffman B.; ''Cdc25A stability is controlled by the ubiquitin-proteasome pathway during cell cycle progression and terminal differentiation.''; PubMedEurope PMCScholia
Das S, Raj L, Zhao B, Kimura Y, Bernstein A, Aaronson SA, Lee SW.; ''Hzf Determines cell survival upon genotoxic stress by modulating p53 transactivation.''; PubMedEurope PMCScholia
Dornan D, Shimizu H, Mah A, Dudhela T, Eby M, O'rourke K, Seshagiri S, Dixit VM.; ''ATM engages autodegradation of the E3 ubiquitin ligase COP1 after DNA damage.''; PubMedEurope PMCScholia
Chaturvedi P, Eng WK, Zhu Y, Mattern MR, Mishra R, Hurle MR, Zhang X, Annan RS, Lu Q, Faucette LF, Scott GF, Li X, Carr SA, Johnson RK, Winkler JD, Zhou BB.; ''Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway.''; PubMedEurope PMCScholia
Hupp TR, Lane DP.; ''Allosteric activation of latent p53 tetramers.''; PubMedEurope PMCScholia
Lovly CM, Yan L, Ryan CE, Takada S, Piwnica-Worms H.; ''Regulation of Chk2 ubiquitination and signaling through autophosphorylation of serine 379.''; PubMedEurope PMCScholia
Bochkareva E, Korolev S, Lees-Miller SP, Bochkarev A.; ''Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA.''; PubMedEurope PMCScholia
While the ATR-ATRIP complex binds only poorly to RPA complexed with ssDNA lengths of 30 or 50 nt, binding is significantly enhanced in the presence of a 75 nt ssDNA molecule. Complex formation is primarily mediated by physical interaction between ATRIP and RPA. Multiple elements within the ATRIP molecule can bind to the RPA-ssDNA complex, including residues 1-107 (highest affinity), 218-390, and 390-791 (lowest afiinity). Although the full-length ATRIP is unable to bind ssDNA, an internal region (108-390) can weakly bind ssDNA when present in rabbit reticulocyte lysates. ATR can bind to the ssDNA directly independent of RPA, but this binding is inhibited by ATRIP. Upon binding, the ATR kinase becomes activated and can directly phosphorylate substrates such as Rad17.
The ATR (ATM- and rad3-related) kinase is an essential checkpoint factor in human cells. In response to replication stress (i.e., stresses that cause replication fork stalling) or ultraviolet radiation, ATR becomes active and phosphorylates numerous factors involved in the checkpoint response including the checkpoint kinase Chk1. ATR is invariably associated with ATRIP (ATR-interacting protein) in human cells. Depletion of ATRIP by siRNA causes a loss of ATR without affecting ATR mRNA levels indicating that complex formation stabilizes ATR. ATRIP is also a substrate for the ATR kinase, but this modification does not play a significant role in the recruitment of ATR-ATRIP to sites of damage, the activation of Chk1, or the modification of p53.
DNA double strand break (DSB) response involves sensing of DNA DSBs by the MRN complex which triggers ATM activation. ATM phosphorylates a number of proteins involved in DNA damage checkpoint signaling, as well as proteins directly involved in the repair of DNA DSBs. For a recent review, please refer to Ciccia and Elledge, 2010.
The molecules that directly interact with Mad1 is unknown. However molecular genetic data has defined an assembly pathway consisting of CENP-I, HEC1, Mps1 that specifies the assembly of Mad1.
The Rad17-RFC complex is a heteropentamer structurally similar to RFC. The Rad17-RFC complex contains the four smaller RFC subunits (Rfc2 [p37], Rfc3 [p36], Rfc4 [p40], and Rfc5 [p38]) and the 75 kDa Rad17 subunit in place of the Rfc1 [p140] subunit. The Rad17 complex contains a weak ATPase that is poorly stimulated by primed DNA. Along with binding the 9-1-1 complex and RPA, the Rad17-RFC complex interacts with human MCM7 protein. Each of these interactions is critical for Chk1 activation.
The Rad17 subunit is conserved evolutionarily with the protein showing 49% identity at the amino acid level with the S. pombe rad17 protein. Targeted deletion of the N-terminal region of mouse Rad17 leads to embryonic lethality, strongly suggesting that human Rad17 is also essential for long-term viability.
The Rad9-Hus1-Rad1 (9-1-1) complex is a ring-shaped heterotrimeric complex. Under genotoxic stress conditions, it can be loaded onto DNA at sites of damage or stalled forks by the Rad17 complex.
RPA associates with ssDNA in distinct complexes that can be distinguished by the length of ssDNA occluded by each RPA molecule. These complexes reflect the progressive association of distinct DNA-binding domains present in the RPA heterotrimeric structure. Binding is coupled to significant conformational changes within RPA that are observable at the microscopic level. Presumably, the different conformations of free and ssDNA-bound RPA allow the protein to selectively interact with factors such as ATR-ATRIP when bound to DNA.
Rad17-RFC complex associates with DNA substrates containing ssDNA regions including gapped or primed DNA in an ATP-independent reaction. Loading of the Rad9-Hus1-Rad1 (9-1-1) complex occurs preferentially on DNA substrates containing a 5' recessed end. This contrasts with the loading of PCNA by RFC which preferentially occurs on DNA with 3' recessed ends.
A major known function of the 9-1-1 complex is to recruit Chk1 to stalled replication forks for activation by ATR. However, the presence of the 9-1-1 complex also alters the ability of Rad17 to become phoshorylated, perhaps suggesting that 9-1-1 may also serve to recruit a subset of ATR substrates. The 9-1-1 complex has also been found to interact with base excision repair factors human DNA polymerase beta, flap endonuclease FEN1, and the S. pombe MutY homolog (SpMYH), indicating that 9-1-1 also plays a direct role in DNA repair.
The association of Mad1 with the kinetochore is the first step in the process of Mad2 mediated amplification of the signal from defective kinetochores.
In vitro structural studies have shown that Mad2 undergoes a major conformational change upon binding to Mad1. This conformational change is postulated to activate Mad2 into a high affinity state which can bind and sequester Cdc20 from the APC.
In the direct inhibition model, association of the MCC with APCC results in the inactivation of APC/C. However, the affinity between MCC and APC/C is not high, so that the inhibition is readily reversible. The role of unattached kinetochores is to sensitize the APC/C to prolonged inhibition by the MCC.
In the sequestration model, the Mad2 molecules that dissociate from unattached kinetochores are perceived to bind to Cdc20, a protein that recruits specific substrates to the APC/C. Consequently, Mad2 indirectly inhibits the APC/C by sequestering its activator, Cdc20. This requires interaction between Mad1 and Mad2. Cdc20 and Mad1 bind to the same site on Mad2.
Upon release from the kinetochore, Mad2 associates with Cdc20, hBUBR1, and hBUB3 to form the Mitotic Checkpoint Complex (MCC). Assembly of this complex does not depend on kinetochores but this complex can only inhibit APC/C that has undergone mitotic modifications.
The mechanism by which the conformationally altered inhibitory form of Mad2 is released from its association with Mad1 at the kinetochore is not known. Mad1 and Cdc20 have a common 10 residue Mad2 binding motif. Therefore, one possibility is that Mad2 is transferred competitively from Mad1 to Cdc20 (Luo et al., 2002; Sironi et al., 2002).
WEE1, a nuclear kinase, phosphorylates cyclin B1:Cdc2 (CCNB1:CDK1) on tyrosine 15 (Y15), inactivating the complex (Parker and Piwnica-Worms 1992, McGowan and Russell 1993). The complex of cyclin B2 and Cdc2 (CCNB2:CDK1) is also phosphorylated on Y15 (Galaktionov and Beach 1991).
The Rad17-RFC complex is involved in an early stage of the genotoxic stress response. The major function of the protein complex is to load the Rad9-Hus1-Rad1 (9-1-1) complex onto DNA at sites of damage and/or stalled replication forks. This reaction is conceptually similar to the loading of the PCNA sliding clamp onto DNA by RFC. The association of the Rad17-RFC complex with ssDNA or gapped or primed DNA is significantly stimulated by RPA, but not by the heterologous E. coli SSB. Loading of the human 9-1-1 complex onto such DNA templates is also strongly stimulated by cognate RPA, but not yeast RPA. Although Rad17 and Rad9 are substrates of the ATR kinase activity, loading of the Rad17 and 9-1-1 complexes onto DNA occurs independent of ATR.
The Rad17-RFC complex is a heteropentamer structurally similar to RFC. The complex contains the four smaller RFC subunits (Rfc2 [p37], Rfc3 [p36], Rfc4 [p40], and Rfc5 [p38]) and the 75 kDa Rad17 subunit in place of the Rfc1 [p140] subunit. The Rad17 complex contains a weak ATPase that is slightly stimulated by primed DNA. Along with binding the 9-1-1 complex and RPA, the Rad17-RFC complex interacts with human MCM7 protein. Each of these interactions is critical for Chk1 activation.
The Rad17 subunit is conserved evolutionarily with the protein showing 49% identity at the amino acid level with the S. pombe rad17 protein. Targeted deletion of the N-terminal region of mouse Rad17 leads to embryonic lethality, strongly suggesting that human Rad17 is also essential for long-term viability.
Rad17-RFC complex associates with DNA substrates containing ssDNA regions including gapped or primed DNA in an ATP-independent reaction. Loading of the Rad9-Hus1-Rad1 (9-1-1) complex occurs preferentially on DNA substrates containing a 5' recessed end. This contrasts with the loading of PCNA by RFC which preferentially occurs on DNA with 3' recessed ends.
Chk1 is a checkpoint kinase activated during genotoxic stress. Like ATR, Chk1 is essential for viability in mammals. Targeted gene disruption in mice shows that loss of Chk1 causes peri-implantation embryonic lethality. Even though ATR-ATRIP not bound to ssDNA can phosphorylate Chk1, Chk1 activation is greatly enhanced when recruited to stalled replication forks by physical interaction with a modified form of claspin and the Rad9-Hus1-Rad1 sliding clamp. Activation of Chk1 occurs following phosphorylation of two sites (serine 317 and serine 345). Mutational analysis indicates that modification of both sites is essential for maximal kinase activity, while phosphorylation of only a single site causes only weak activation of Chk1. Following phosphorylation, Chk1 can diffuse away from the complex to further amplify the checkpoint signal. ATR appears to be the primary kinase activating Chk1 as conditions that activate ATR (ultraviolet irradiation or treatment with hydroxyurea) also activate Chk1. Stresses that activate ATM, e.g., ionizing irradiation, do not cause significant Chk1 activation. While the ATR and ATM pathways are distinct, there is interplay between the two. For example, double-strand DNA breaks can be processed in an ATM-dependent manner to generate structures that can cause ATR and hence Chk1 activation. The ATR and ATM pathways also have mechanistic similarities. Analogous to the Chk1 kinase existing downstream of ATR, the Chk2 checkpoint kinase is modified and activated by ATM. Although having distinct structures, Chk1 and Chk2 also have overlapping targets with some substrate sites phosphorylatable by both kinases (e.g., serine 20 of p53).
When a DNA replication fork encounters DNA lesions (e.g., cyclobutane pyrimidine dimers or alkylated bases) stalling of the replicative DNA polymerase may occur. This can lead to dissociation or 'uncoupling' of the DNA polymerase from the DNA helicase and generation of long regions of persistent ssDNA. Uncoupling can also occur in response to other genotoxic stresses such as reduced dNTP pools caused by hydroxyurea treatment which inhibits cellular ribonucleotide diphosphate reductase. The exposed ssDNA is bound by the single-stranded DNA binding protein RPA. The persistent nature of this RPA-ssDNA complex (as opposed to a more-transient complex found at an active replication fork) allows it to serve as a signal for replication stress that can be recognized by the ATR-ATRIP and Rad17-Rfc2-5 complexes.
RPA associates with ssDNA in distinct complexes that can be distinguished by the length of ssDNA occluded by each RPA molecule. These complexes reflect the progressive association of distinct DNA-binding domains present in the RPA heterotrimeric structure. Binding is coupled to significant conformational changes within RPA that are observable at the microscopic level. Presumably, the different conformations of free and ssDNA-bound RPA allow the protein to selectively interact with factors such as ATR-ATRIP when bound to DNA.
ATR kinase activity is stimulated upon binding of the ATR-ATRIP complex to an RPA-ssDNA complex. ATR can subsequently phosphorylate and activate the checkpoint kinase Chk1, allowing further amplification of the checkpoint signal. The ATR and Chk1 kinases then modify a variety of factors that can lead to stabilization of stalled DNA replication forks, inhibition of origin firing, inhibition of cell cycle progression, mobilization of DNA repair factors, and induction of apoptosis. This checkpoint signaling mechanism is highly conserved in eukaryotes, and homologues of ATR and ATRIP are found in such organisms as S. cerevisiae (Mec1 and Ddc2, respectively), S. pombe (rad3 and rad26, respectively), and X. laevis (Xatr and Xatrip, respectively).
The ATR (ATM- and rad3-related) kinase is an essential checkpoint factor in human cells. In response to replication stress (i.e., stresses that cause replication fork stalling) or ultraviolet radiation, ATR becomes active and phosphorylates numerous factors involved in the checkpoint response including the checkpoint kinase Chk1. ATR is invariably associated with ATRIP (ATR-interacting protein) in human cells. Depletion of ATRIP by siRNA causes a loss of ATR protein without affecting ATR mRNA levels indicating that complex formation stabilizes the ATR protein. ATRIP is also a substrate for the ATR kinase, but modification of ATRIP does not significantly regulate the recruitment of ATR-ATRIP to sites of damage, the activation of Chk1, or the modification of p53.
While the ATR-ATRIP complex binds only poorly to RPA complexed with ssDNA lengths of 30 or 50 nt, binding is significantly enhanced in the presence of a 75 nt ssDNA molecule. Complex formation is primarily mediated by physical interaction between ATRIP and RPA. Multiple elements within the ATRIP molecule can bind to the RPA-ssDNA complex, including residues 1-107 (highest affinity), 218-390, and 390-791 (lowest affinity). Although the full-length ATRIP is unable to bind ssDNA, an internal region (108-390) can weakly bind ssDNA when present in rabbit reticulocyte lysates. ATR can bind to the ssDNA directly independent of RPA, but this binding is inhibited by ATRIP. Upon binding, the ATR kinase becomes activated and can directly phosphorylate substrates such as Rad17.
The 9-1-1 complex is a heterotrimeric ring-shaped structure that is loaded onto DNA by the Rad17-RFC complex. In vitro studies indicate that loading is preferred onto DNA substrates containing ssDNA gaps that presumably resemble structures found upon replication fork stalling and DNA polymerase/helicase uncoupling. The Rad17-RFC and 9-1-1 complexes are structurally similar to the RFC (replication factor C) clamp loader and PCNA sliding clamp, respectively, and similar mechanisms are thought to be used during loading of the 9-1-1 complex and PCNA. Upon loading, the 9-1-1 complex can recruit Chk1 onto sites of replication fork uncoupling or DNA damage.
The purified Rad17 and Rad9-Hus1-Rad1 (9-1-1) complexes can form a stable co-complex in the presence of ATP, using Rad17-Rad9 interactions. From computer modeling studies, the Rad17 subunit of the complex is also proposed to interact with the C-terminus of Rad1, p36 with the C-terminus of Hus1, and p38 with the C-terminus of Rad9. A major known function of the 9-1-1 complex is to recruit Chk1 to stalled replication forks for activation by ATR. However, the presence of the 9-1-1 complex also alters the ability of Rad17 to become phosphorylated, perhaps suggesting that 9-1-1 may regulate the recruiment of additional ATR substrates. The 9-1-1 complex has also been found to interact with base excision repair factors human DNA polymerase beta, flap endonuclease FEN1, and the S. pombe MutY homolog (SpMYH), indicating that 9-1-1 also plays a direct role in DNA repair.
Claspin is a replication fork-associated protein important for Chk1 activation. Claspin loads onto the fork during replication origin firing and travels with the fork during DNA synthesis. Upon fork uncoupling and ATR-ATRIP binding to persistent ssDNA, the activated ATR kinase phosphorylates claspin at two primary sites. Modification increases the affinity of claspin for Chk1. Studies of human or Xenopus claspin indicate that phosphorylation of both sites is essential for significant claspin-Chk1 association. Following claspin modification by ATR, Chk1 can be transiently recruited to the stalled replication fork for subsequent phosphorylation and activation by ATR. Activation of Chk1 allows modification of additional downstream targets, thus amplifying the checkpoint signal. While much of the mechanistic information concerning claspin action has been obtained using Xenopus laevis egg extracts and Xenopus claspin, factors with similar activity have been found in various eukaryotic species including S. cerevisiae (MRC1), S. pombe (mrc1), and humans.
Activated ATR phosphorylates human claspin on two sites, threonine 916 and serine 945.
Claspin is loaded onto DNA replication origins during replication initiation. Studies in Xenopus egg extracts indicate claspin loading requires the presence of Cdc45, a factor that promotes the initial unwinding of the origin DNA in the presence of Cdk2. This step is followed by RPA binding which is a prerequisite for recruitment of PCNA and DNA polymerases alpha and delta. As RPA is not required for claspin binding, it is postulated that claspin binds at the time of initial origin unwinding but prior to the initiation of DNA synthesis. Claspin would then continue to associate with replication fork machinery where it can serve as a checkpoint sensor protein. Even though associated with the replication fork, claspin is not an essential DNA replication factor.
Studies of Xenopus claspin indicate that it can physically associate with cognate Cdc45, DNA polymerase epsilon, RPA, RFC, and Rad17-RFC on chromatin. Studies of purified human claspin indicate that it binds with high affinity to branched (or forked) DNA structures that resemble stalled replication forks. Electron microscopy of these complexes indicates that claspin binds as a ring-like structure near the branch. The protein is hypothesized to encircle the DNA at these sites.
During G1, the activity of cyclin-dependent kinases (CDKs) is controlled by the CDK inhibitors (CKIs) CDKN1A (p21) and CDKN1B (p27), thereby preventing premature entry into S phase (Guardavaccaro and Pagano, 2006).
ATM phosphorylation promotes autoubiquitination of COP1 in vitro (Dornan et al., 2006). The number of ubiquitin molecules shown in this reaction is set arbitrarily at 4.
Autoubiquitinated COP1 is degraded by the proteasome. The number of ubiquitin molecules shown in this reaction is arbitrarily set at 4. (Dornan et al., 2006).
CHEK2 (Chk2) kinase is required for phosphorylation of MDM4 at serine residues S342 and S367 in vivo. CHEK2-mediated phosphorylation stimulates MDM4 ubiquitination by MDM2 and subsequent degradation (Chen et al. 2005).
Human MDM4 (MDMX) is phosphorylated on serine residue S403 by ATM. This site is important for MDM2-mediated ubiquitination of MDM4 after induction of double strand DNA breaks (Pereg et al. 2005, Chen et al. 2005).
The N-terminal portion of MDM2 binds the N-terminal transactivation domain of TP53 (p53) and inhibits transcriptional transactivation by TP53 (Momand et al. 1992, Oliner et al. 1992, Oliner et al. 1993, Chen et al. 1993).
CHEK2 (CHK2, Cds1) is recruited to DNA double strand breaks (DSBs) mainly through its interaction with TP53BP1 (53BP1) (Wang et al. 2002), but BRCA1 also contributes to CHEK2 recruitment (Wilson and Stern 2008).
ATM-mediated phosphorylation of CHEK2 (CHK2, Cds1) on threonine residue T68 promotes formation of transitional CHEK2 homodimers primarily through intermolecular interactions of FHA domains and phospho-T68 residues of two CHEK2 protomers (Cai et al. 2009).
Upon dimerization, p-T68-CHEK2 protomers trans-autophosphorylate on serine residue S379 (Lovly et al. 2008) and threonine residues T383 and T387 (Lee et al. 2001). Autophosphorylation leads to dissociation of CHEK2 dimers into active CHEK2 monomers (Cai et al. 2009).
In response to DNA double strand breaks, serine at position 15 of the TP53 (p53) tumor suppressor protein is rapidly phosphorylated by the ATM kinase. This serves to stabilize the p53 protein. A rise in the levels of the p53 protein induces the expression of p21 cyclin-dependent kinase inhibitor. This prevents the normal progression from G1 to S phase, thus providing a check on replication of damaged DNA (Banin et al. 1998, Canman et al. 1998, Khanna et al. 1998).
Upon MDM2-mediated ubiquitination, TP53 is exported from the nucleus to the cytosol. TP53 nuclear export requires the nuclear export sequence (NES) of TP53, but not the NES of MDM2 (Boyd et al. 2000. Geyer et al. 2000).
CHEK1, activated by ATR-mediated phosphorylation, can phosphorylate TP53 at serine residue S20, resulting in the increased half-life of TP53 (Shieh et al. 2000).
ATR, bound to DNA damage sites, phosphorylates TP53 (p53) at serine residue S15. S15 phosphorylation stabilizes TP53 by inhibiting the binding of TP53 to the ubiquitin ligase MDM2 (Tibbetts et al. 1999, Lakin et al. 1999).
Binding of TP53 (p53) to its response elements in the promoter of the CDKN1A (p21) gene stimulates CDKN1A transcription (El-Deiry et al. 1993). Binding of ZNF385A (HZF) to the DNA binding domain of TP53 facilitates CDKN1A induction and the consequent cell cycle arrest (Das et al. 2007).
PCBP4 binding to the 3'-UTR of the CDKN1A (p21) mRNA reduces half-life of the CDKN1A mRNA and the amount of CDKN1A protein. Upon DNA damage, TP53-mediated induction of CDKN1A is rapid, while the induction of PCBP4 is more gradual. It is hypothesized that, under prolonged stress, PCBP4-mediated down-regulation of CDKN1A may switch from G1 cell cycle arrest to G2 arrest, which may precede apoptosis (Scoumanne et al. 2011).
ZNF385A (HZF) forms a complex with TP53 (p53), interacting with the DNA binding domain of TP53. The complex of TP53 and ZNF385A associates with p53 response elements of cell cycle arrest genes, such as CDKN1A (p21) and stimulates their transcription. Under prolonged stress, ZNF385A undergoes ubiquitination and proteasome-mediated degradation, which coincides with expression of TP53-regulated pro-apoptotic genes (Das et al. 2007).
TP53 (p53) binds at least two p53 response elements in the promoter of the CDKN1A (p21, WAF1) gene (El-Deiry et al. 1993, Espinosa et al. 2003). Formation of the complex of TP53 and ZNF385A (HZF) facilitates binding of TP53 to the CDKN1A promoter (Das et al. 2007).
Once MDM4 is phosphorylated by ATM and CHEK2 in response to DNA damage, MDM2 ubiquitinates MDM4 and targets it for degradation (Chen et al. 2005, Pereg et al. 2005). The presence of MDM4 stimulates auto-ubiquitination of MDM2 (Linares et al. 2003).
To efficiently function as an E3 ubiquitin ligase, MDM2 has to form dimers or higher order oligomers. MDM2 can homodimerize (Cheng et al. 2011) or heterodimerize with MDM4 (MDMX) (Sharp et al. 1999, Huang et al. 2011, Pant et al. 2011). Dimerization involves the RING domain of MDM2 and/or MDM4. Heterodimers of MDM2 and MDM4 may be particularly important during embryonic development (Pant et al. 2011).
MDM2 is an ubiquitin ligase whose expression is positively regulated by TP53 (p53) (Wu et al. 1993). MDM2 binds TP53 tetramer (Maki 1999) and promotes its ubiquitination and subsequent degradation (Fuchs et al. 1998). Formation of MDM2 homodimers (Cheng et al. 2011) or heterodimers with MDM4 (MDMX) is needed for efficient ubiquitination of TP53 (Linares et al. 2003). While MDM2-TP53 binding occurs at the amino-terminus of TP53, MDM2 ubiquitinates TP53 lysine residues at the carboxy-terminus. Acetylation of those lysines can inhibit MDM2-dependent ubiquitination (Li et al. 2002).
ATM phosphorylates MDM2 on three serine residues (S386, S395, S407) and one threonine residue (T419) in vicinity to the RING domain. ATM-mediated phosphorylation of MDM2 in response to DNA damage (DNA double strand breaks) prevents MDM2 dimerization, binding of TP53 (p53) and MDM2-mediated ubiquitination of TP53 (Cheng et al. 2009, Cheng et al. 2011).
During G1, the activity of cyclin-dependent kinases (CDKs) is controlled by the CDK inhibitors (CKIs) CDKN1A (p21) and CDKN1B (p27), thereby preventing premature entry into S phase (see Guardavaccaro and Pagano, 2006). The efficient recognition and ubiquitination of p27 by the SCF (Skp2) complex requires the formation of a trimeric complex containing p27 and cyclin E/A:Cdk2.
At the beginning of this reaction, 1 molecule of 'ubiquitin', and 1 molecule of 'phospho-Cdc25A' are present. At the end of this reaction, 1 molecule of 'Ubiquitinated Phospho-Cdc25A' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'ubiquitin-protein ligase activity' of 'Ubiquitin ligase' (Bernardi et al. 2000).
At the beginning of this reaction, 1 molecule of 'Ubiquitinated Phospho-Cdc25A' is present. At the end of this reaction, 1 molecule of 'Amino Acid' is present.
This reaction takes place in the 'cytosol' and is mediated by the 'endopeptidase activity' of '26S proteasome' (Bernardi et al. 2000).
CHEK2 (Chk2) phosphorylates TP53 (p53) at serine residue S20 (Hirao et al. 2000, Shieh et al. 2000, Chehab et al. 2000). Phosphorylation of TP53 at serine residue S20 is necessary for DNA damage-induced TP53 stabilization as it compromises the interaction of TP53 with the ubiquitin ligase MDM2 (Chehab et al. 1999, Chehab et al. 2000). S20 phosphorylation is also required for the induction of TP53-dependent transcripts in response to DNA damage (Hirao et al. 2000).
Activated ATM phosphorylates CHEK2 (CHK2, Cds1) on threonine residue T68 (Matsuoka et al. 2000, Melchionna et al. 2000). The presence of BRCA1 and TP53BP1 positively regulates ATM-mediated phosphorylation of CHEK2 (Wang et al. 2002, Foray et al. 2003). ATM-mediated phosphorylation causes formation of CHEK2 dimers and dissociation of CHEK2 from chromatin (Li and Stern 2005).
Phosphorylation of Cdc25C at Ser 216 results in both the inhibition of Cdc25C phosphatase activity and the creation of a 14-3-3 docking site (Peng et al. 1997).
CDC25C is phosphorylated by CHK1 at ser-216 (Blasina et al.,1999 ) resulting in both inhibition of the CDC25 phosphatase activity and creation of a 14-3-3 docking site (Peng et al., 1997). Association of 14-3-3 proteins with phosphorylated CDC25C (p-S216-CDC25C) is thought to result in retention of this complex within the cytoplasm (Dalal et al., 1999; Graves et al, 2001).
Cdc25C is negatively regulated by phosphorylation on Ser 216, the 14-3-3-binding site. This is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage (Chaturvedi et al, 1999; Bulavin et al., 2003).
Since MDM2-mediated ubiquitination of TP53 promotes translocation of TP53 to the cytosol, and since GTSE1-facilitated translocation of TP53 to the cytosol depends on the functional MDM2 (with no reported interaction between GTSE1 and MDM2) (Monte et al. 2004), it is plausible that GTSE1 binds to TP53 polyubiquitinated by MDM2. The interaction between TP53 and GTSE1 involves the C-terminal regulatory domain of TP53 and the C-terminus of GTSE1 (Monte et al. 2003).
Binding of GTSE1 to TP53 (p53) in the nucleus promotes translocation of TP53 to the cytosol. This process is dependent on the nuclear export signal (NES) of GTSE1 (Monte et al. 2004).
GTSE1 promotes down-regulation of TP53 in a proteasome-dependent way. Nuclear export of TP53 facilitated by GTSE1 and MDM2likely makes ubiquitinated TP53 available to the proteasome machinery. GTSE1-mediated decrease of TP53 levels is needed for the G2 checkpoint recovery (cell cycle re-entry after DNA damage induced G2 arrest) and rescues cells from DNA damage induced apoptosis during S/G2 phase (Monte et al. 2003, Monte et al. 2004).
Phosphorylated CDC25C translocates to the cytoplasm. Phosphorylation at serine residue S216 is not a prerequisite for translocation to the cytoplasm (Dalal et al., 1999).
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DataNodes
overhanging
ssDNA-DSBs:p-MRN:p-S1981,Ac-K3016-ATM:KAT5:BRCA1-C complex:EXO1,DNA2:BLM,WRN:p-S990,Ac-K1249-BRIP1:RAD17:RFC:RAD9:HUS1:RAD1:RHNO1:TOPBP1pre-replicative
complex:CDC45A:Cdk2:p21/p27
complexDNA
DSBs:p-MRN:p-S1981,Ac-K3016-ATM:KAT5:K63PolyUb-K14,K16,p-S139-H2AFX,Me2K21-HIST1H4A-Nucleosome:p-5T-MDC1:p-S102-WHSC1:RNF8:Zn2+:SUMO1:p-T4827-HERC2:UBE2N:UBE2V2:RNF168:PIAS4:p-S25,S1778-TP53BP1:p-4S,2T-BRCA1-A complex:CHEK2DNA
DSBs:p-MRN:p-S1981,Ac-K3016-ATM:KAT5:K63PolyUb-K14,K16,p-S139-H2AFX,Me2K21-HIST1H4A-Nucleosome:p-5T-MDC1:p-S102-WHSC1:RNF8:Zn2+:SUMO1:p-T4827-HERC2:UBE2N:UBE2V2:RNF168:PIAS4:p-S25,S1778-TP53BP1:p-4S,2T-BRCA1-A complexThe Rad17 subunit is conserved evolutionarily with the protein showing 49% identity at the amino acid level with the S. pombe rad17 protein. Targeted deletion of the N-terminal region of mouse Rad17 leads to embryonic lethality, strongly suggesting that human Rad17 is also essential for long-term viability.
dimer,
p-S166,S188-MDM2,MDM4:TP53dimer,
p-S166,S188-MDM2:MDM4anaphase promoting
complex (APC/C)Annotated Interactions
overhanging
ssDNA-DSBs:p-MRN:p-S1981,Ac-K3016-ATM:KAT5:BRCA1-C complex:EXO1,DNA2:BLM,WRN:p-S990,Ac-K1249-BRIP1:RAD17:RFC:RAD9:HUS1:RAD1:RHNO1:TOPBP1pre-replicative
complex:CDC45A:Cdk2:p21/p27
complexDNA
DSBs:p-MRN:p-S1981,Ac-K3016-ATM:KAT5:K63PolyUb-K14,K16,p-S139-H2AFX,Me2K21-HIST1H4A-Nucleosome:p-5T-MDC1:p-S102-WHSC1:RNF8:Zn2+:SUMO1:p-T4827-HERC2:UBE2N:UBE2V2:RNF168:PIAS4:p-S25,S1778-TP53BP1:p-4S,2T-BRCA1-A complex:CHEK2DNA
DSBs:p-MRN:p-S1981,Ac-K3016-ATM:KAT5:K63PolyUb-K14,K16,p-S139-H2AFX,Me2K21-HIST1H4A-Nucleosome:p-5T-MDC1:p-S102-WHSC1:RNF8:Zn2+:SUMO1:p-T4827-HERC2:UBE2N:UBE2V2:RNF168:PIAS4:p-S25,S1778-TP53BP1:p-4S,2T-BRCA1-A complex:CHEK2DNA
DSBs:p-MRN:p-S1981,Ac-K3016-ATM:KAT5:K63PolyUb-K14,K16,p-S139-H2AFX,Me2K21-HIST1H4A-Nucleosome:p-5T-MDC1:p-S102-WHSC1:RNF8:Zn2+:SUMO1:p-T4827-HERC2:UBE2N:UBE2V2:RNF168:PIAS4:p-S25,S1778-TP53BP1:p-4S,2T-BRCA1-A complex:CHEK2DNA
DSBs:p-MRN:p-S1981,Ac-K3016-ATM:KAT5:K63PolyUb-K14,K16,p-S139-H2AFX,Me2K21-HIST1H4A-Nucleosome:p-5T-MDC1:p-S102-WHSC1:RNF8:Zn2+:SUMO1:p-T4827-HERC2:UBE2N:UBE2V2:RNF168:PIAS4:p-S25,S1778-TP53BP1:p-4S,2T-BRCA1-A complexDNA
DSBs:p-MRN:p-S1981,Ac-K3016-ATM:KAT5:K63PolyUb-K14,K16,p-S139-H2AFX,Me2K21-HIST1H4A-Nucleosome:p-5T-MDC1:p-S102-WHSC1:RNF8:Zn2+:SUMO1:p-T4827-HERC2:UBE2N:UBE2V2:RNF168:PIAS4:p-S25,S1778-TP53BP1:p-4S,2T-BRCA1-A complexThe Rad17-RFC complex is a heteropentamer structurally similar to RFC. The complex contains the four smaller RFC subunits (Rfc2 [p37], Rfc3 [p36], Rfc4 [p40], and Rfc5 [p38]) and the 75 kDa Rad17 subunit in place of the Rfc1 [p140] subunit. The Rad17 complex contains a weak ATPase that is slightly stimulated by primed DNA. Along with binding the 9-1-1 complex and RPA, the Rad17-RFC complex interacts with human MCM7 protein. Each of these interactions is critical for Chk1 activation.
The Rad17 subunit is conserved evolutionarily with the protein showing 49% identity at the amino acid level with the S. pombe rad17 protein. Targeted deletion of the N-terminal region of mouse Rad17 leads to embryonic lethality, strongly suggesting that human Rad17 is also essential for long-term viability.
Rad17-RFC complex associates with DNA substrates containing ssDNA regions including gapped or primed DNA in an ATP-independent reaction. Loading of the Rad9-Hus1-Rad1 (9-1-1) complex occurs preferentially on DNA substrates containing a 5' recessed end. This contrasts with the loading of PCNA by RFC which preferentially occurs on DNA with 3' recessed ends.
RPA associates with ssDNA in distinct complexes that can be distinguished by the length of ssDNA occluded by each RPA molecule. These complexes reflect the progressive association of distinct DNA-binding domains present in the RPA heterotrimeric structure. Binding is coupled to significant conformational changes within RPA that are observable at the microscopic level. Presumably, the different conformations of free and ssDNA-bound RPA allow the protein to selectively interact with factors such as ATR-ATRIP when bound to DNA.
The ATR (ATM- and rad3-related) kinase is an essential checkpoint factor in human cells. In response to replication stress (i.e., stresses that cause replication fork stalling) or ultraviolet radiation, ATR becomes active and phosphorylates numerous factors involved in the checkpoint response including the checkpoint kinase Chk1. ATR is invariably associated with ATRIP (ATR-interacting protein) in human cells. Depletion of ATRIP by siRNA causes a loss of ATR protein without affecting ATR mRNA levels indicating that complex formation stabilizes the ATR protein. ATRIP is also a substrate for the ATR kinase, but modification of ATRIP does not significantly regulate the recruitment of ATR-ATRIP to sites of damage, the activation of Chk1, or the modification of p53.
While the ATR-ATRIP complex binds only poorly to RPA complexed with ssDNA lengths of 30 or 50 nt, binding is significantly enhanced in the presence of a 75 nt ssDNA molecule. Complex formation is primarily mediated by physical interaction between ATRIP and RPA. Multiple elements within the ATRIP molecule can bind to the RPA-ssDNA complex, including residues 1-107 (highest affinity), 218-390, and 390-791 (lowest affinity). Although the full-length ATRIP is unable to bind ssDNA, an internal region (108-390) can weakly bind ssDNA when present in rabbit reticulocyte lysates. ATR can bind to the ssDNA directly independent of RPA, but this binding is inhibited by ATRIP. Upon binding, the ATR kinase becomes activated and can directly phosphorylate substrates such as Rad17.
The purified Rad17 and Rad9-Hus1-Rad1 (9-1-1) complexes can form a stable co-complex in the presence of ATP, using Rad17-Rad9 interactions. From computer modeling studies, the Rad17 subunit of the complex is also proposed to interact with the C-terminus of Rad1, p36 with the C-terminus of Hus1, and p38 with the C-terminus of Rad9. A major known function of the 9-1-1 complex is to recruit Chk1 to stalled replication forks for activation by ATR. However, the presence of the 9-1-1 complex also alters the ability of Rad17 to become phosphorylated, perhaps suggesting that 9-1-1 may regulate the recruiment of additional ATR substrates. The 9-1-1 complex has also been found to interact with base excision repair factors human DNA polymerase beta, flap endonuclease FEN1, and the S. pombe MutY homolog (SpMYH), indicating that 9-1-1 also plays a direct role in DNA repair.
Activated ATR phosphorylates human claspin on two sites, threonine 916 and serine 945.
Studies of Xenopus claspin indicate that it can physically associate with cognate Cdc45, DNA polymerase epsilon, RPA, RFC, and Rad17-RFC on chromatin. Studies of purified human claspin indicate that it binds with high affinity to branched (or forked) DNA structures that resemble stalled replication forks. Electron microscopy of these complexes indicates that claspin binds as a ring-like structure near the branch. The protein is hypothesized to encircle the DNA at these sites.
This reaction takes place in the 'cytosol' and is mediated by the 'ubiquitin-protein ligase activity' of 'Ubiquitin ligase' (Bernardi et al. 2000).
This reaction takes place in the 'cytosol' and is mediated by the 'endopeptidase activity' of '26S proteasome' (Bernardi et al. 2000).
dimer,
p-S166,S188-MDM2,MDM4:TP53dimer,
p-S166,S188-MDM2,MDM4:TP53dimer,
p-S166,S188-MDM2,MDM4:TP53dimer,
p-S166,S188-MDM2:MDM4dimer,
p-S166,S188-MDM2:MDM4dimer,
p-S166,S188-MDM2:MDM4anaphase promoting
complex (APC/C)