Die Bindung von PAR an Chk1 steuert die Entwicklung und die Gewebeaufrechterhaltung
Zusammenfassung der Projektergebnisse
DNA replication ensures faithful replication of genomic information that pass-through generations of cells and organism and therefore is essential for a life in order to produce progenies. Thus cells evolve a number of cellular mechanisms including the DNA damage response (DDR) to safeguard the fitness and fidelity of DNA replication. The ATR-Chk1 pathway is activated in response to any damage or stress to replication forks and thereby maintains the replication fork stability and cell viability. The homeostasis of the poly(ADP-ribose) (PAR) metabolism plays an important role in the repair of stalled replication forks and PARP inhibitors have been developed as anti-cancer drugs for its synthetic killing in DNA repair-deficient human cancers. We identified a PAR binding regulatory (PbR) motif in Chk1, a mutation of which compromises the Chk1 activation in response to replication fork poisoning. Due to the failure of our several attempts to generate PbR-Chk1 mutant mice, we could not study the importance of PbR-Chk1 mutation during embryonic development, adult tissue homeostasis and the development of pathogenesis, including cancer. Alternatively, we established embryonic stem (ES) cells and somatic cell models (fibroblasts), which carry PbR- Chk1 mutation, and carried out following studies: (1) To study the biological response of PAR binding to Chk1 in various DNA damage responses. PARylation is a dynamic cellular response to stress and its product PAR serves as a scaffold platform to help other partners exert their function, for example in DNA repair and cell cycle checkpoint. To separate the scaffold function of PARP1 from its enzymatic activity, which produces PAR, we generated a hypoPARylation mouse model in which the kinetics of the PARylation and the PAR chain complexity are compromised. Hypo-PARylation compromised the DNA replication stall-induced DDR, leading to senescence and cell death, indicating that the homeostasis and kinetics of PARylation is important for the cell fate after DNA damage. We attempted to generate a mouse model in which the PAR binding to Chk1 is disrupted, but failed due to the splicing bias of the targeted Chk1 locus leading to a complete deletion of Chk1. We alternatively generated a PbR-knockin ES cell model to study the biological function of PbR in cell cycle checkpoint and cell lethality. We found that the PbR mutation supports the functions of Chk1 and the S-phase checkpoint under physiological conditions, but rendered cells sensitive to replication poison hydroxyurea (HU) and caused an accumulation of DNA damage due to stalled DNA replication. ATR inhibitor (ATRi) further sensitizes PbR-Chk1 ES cells to HU-induced replication stress. Our PbR-Chk1 ES cell model represents the first separation-of-function mutation of Chk1, allowing to study its functions at stalled replication forks, most likely independent from its key function in S-phase checkpoint. (2) To identify partners that cooperate with PbR-Chk1 in synthetic lethality. PARP1 inhibitor has been now used in clinics to kill cancer cells that are deficient in DNA repair pathways of replication damage via an approach called synthetic lethality. In BRCA1 proficient cells, stalled replication forks can be repaired by two pathways: PARP1-mediated SSB repair and HRR (e.g., via Rad51). Chk1 is a key regulator of the S-phase checkpoint; however, Chk1 inhibitors (Chk1i) has not been successfully developed as anti-cancer drugs because of its toxicity to normal cells. We therefore decided to search for molecular pathways that cooperate with PAR binding to Chk1 as an alternative strategy to treat cancer. To search for potential candidates that would cooperate with PbR-Chk1 in synthetic lethality, we employed a largescale genomic screening of 130000 genes using CRISPR/Cas9 technology and obtained many interesting hits that exhibit synthetic lethality. Naturally these hits will be verified molecularly and functionally, for example, by knocking down of these hits in PbR-Chk1 mutant cells and analysing their survival. Some of the hits will be explored for the molecular mechanisms which cooperate with PARP1 activity, PAR formation and PAR-Chk1 mediated S phase checkpoint. The pro-survival function of the hits will be evaluated in counter against the cell toxicity of other drugs. This information is very useful for future development if these pathways can be targeted for cancer cell killing.
Projektbezogene Publikationen (Auswahl)
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Kinetics of poly(ADP-ribosyl)ation, but not PARP1 itself, determines the cell fate in response to DNA damage in vitro and in vivo. Nucleic Acids Res. 45(19):11174-11192 (2017)
Schuhwerk, H., Bruhn, C., Siniuk, K., Min, W., Erener, S., Grigaravicius, P., Krüger, A., Ferrari, E., Zubel, T., Lazaro, D., Monajembashi, S., Kiesow, K., Kroll, T., Bürkle, A., Mangerich, A., Hottiger, M. & Wang, Z.-Q.
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PARPing for Balance in the Homeostasis of Poly(ADP-ribosyl)ation. Seminar Cell Dev Biol 63:81-91 (2017)
Schuhwerk, H., Atteya, R., Siniuk, K. & Wang, Z.-Q.
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XRCC1 Mutation is Associated with PARP1 Hyperactivation and Cerebellar Ataxia. Nature 541(7635):87-91 (2017)
Hoch, N., Hana, H., Stuart, R., Martine, T., Emilia, K., Ju. L., Zeng, Z., McKinnon, P., Wang, Z.-Q., Wagner, J., Yoon, G., Hornyak, P., Gittens, W., Rey, S., Staras, K., Mancini, G. & Caldecott, K.
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The Enigmatic Function of PARP1: From PARylation Activity to PAR Readers. Cells (2019)
Kamaletdinova, T., Fanaei-Kahrani, Z, & Wang, Z.-Q.
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ADP-ribosyltransferases, an update on function and nomenclature. FEBS J. 2021 Jul 29
Lüscher B, Ahel I, Altmeyer M, Ashworth A, Bai P, Chang P, Cohen M, Corda D, Dantzer F, Daugherty MD, Dawson TM, Dawson VL, Deindl S, Fehr AR, Feijs KLH, Filippov DV, Gagné JP, Grimaldi G, Guettler S, Hoch NC, Hottiger MO, Korn P, Kraus WL, Ladurner A, Lehtiö L, Leung AKL, Lord CJ, Mangerich A, Matic I, Matthews J, Moldovan GL, Moss J, Natoli G, Nielsen ML, Niepel M, Nolte F, Pascal J, Paschal BM, Pawłowski K, Poirier GG, Smith S, Timinszky G, Wang Z.-Q., Yélamos J, Yu X, Zaja R, Ziegler M
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Poly(ADP-ribose) regulation in cell cycle control and cancer therapy
Kanstantsin Siuniuk
