PAR binding to Chk1 regulates development and tissue homeostasis

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Single-stranded DNA breaks (SSBs) and replication stress activate PARP1 that catalyses the formation of poly(ADP-ribose) (PAR) polymers as well as the PARylation of target proteins. PAR has been hypothesised to function as a signal to other cellular targets, including repair proteins. PARylation and PARP1 have been shown to be important for multiple cellular processes, including DNA repair, chromatin modulation, transcription, apoptosis, inflammation and ageing. SSBs and replication poison also trigger the ATR-Chk1 DNA damage response (DDR) pathway. Chk1 phosphorylation by ATR is a key event in the S-phase checkpoint. ATR can be PARylated in the S-phase checkpoint and the PARP inhibitor can over-activate Chk1 leading to the S-phase arrest. These observations suggest a role of PARylation in the ATR-Chk1-mediated S-phase checkpoint control. We recently identified a novel PAR-binding motif (PbR) in Chk1 and found interplay between the PAR metabolism and cell cycle progression control. While long chains of PAR stimulate the Chk1 kinase activity, short chains of PAR repress this stimulation, implying a dynamic fine-tuning of the S-phase checkpoint by PAR homeostasis. We also found that a PbR mutation sensitises BRCA1 mutant cancer cells to cell death, which indicates a synergistic killing of breast cancer cells. However, the biological significance and the molecular mechanisms of PAR binding to Chk1 in vivo are largely unknown. Thus, we propose to carry out the following studies:(1) To define the interactive roles of PAR and Chk1 in the ATR-Chk1 pathway in response to SSB and replication. (2) To generate the PbR knockin mouse model to investigate the biological significance of the PbR motif of Chk1 in cells and mice for viability, embryonic and postnatal development and tissue homeostasis. We will engineer knock-in mice that carry the mutant PbR-Chk1 (PbRC/A-Chk1) to dissect the biological function of PAR binding to Chk1 using genetics, cellular and molecular tools. We will analyse the general and neuro-development of the PbRC/A-Chk1 mutant mice to examine whether these mice exhibit a high level of replicative stress during embryogenesis or postnatal life, or accelerated ageing, which may mimic the Seckel phenotype of ATR hypomorphic mice. The proposed studies will delineate the biological function of the network of ATR-Chk1 and PARP1/PAR in handling DNA damage signals in vivo. Exploring the specific function of the PAR metabolism in the ATR-Chk1 activation may lead to new therapeutic strategies for the treatment of human malignancy.