Abstract
The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease exhibit DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer. Here, we provide a body of evidence suggesting that SPRTN activates the ATR-CHK1 phosphorylation signalling cascade during physiological DNA replication by proteolysis-dependent eviction of CHK1 from replicative chromatin. During this process, SPRTN proteolyses the C-terminal/inhibitory part of CHK1, liberating N-terminal CHK1 kinase active fragments. Simultaneously, CHK1 full length and its N-terminal fragments phosphorylate SPRTN at the C-terminal regulatory domain, which stimulates SPRTN recruitment to chromatin to promote unperturbed DNA replication fork progression and DPC repair. Our data suggest that a SPRTN-CHK1 cross-activation loop plays a part in DNA replication and protection from DNA replication stress. Finally, our results with purified components of this pathway further support the proposed model of a SPRTN-CHK1 cross-activation loop.
Highlights
The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells
The main stimulus for ATR-CHK1 activation is replication protein A (RPA)-coated single-stranded DNA that typically forms upon DNA replication fork stalling due to uncoupling of the cell division cycle 45 (Cdc45), minichromosome maintenance protein complex 2-7 (Mcm2-7) and go-ichi-ni-san protein complex GINS (CMG) helicase complex from DNA polymerases[5]
Analysis of Ruijs–Aalfs Syndrome (RJALS) patient and SPRTN-depleted cells revealed that SPRTN protease activity is essential for DNA replication fork progression, cell cycle progression and G2/M checkpoint activation after DNA replication stress but not ionising radiation[25,28,31]
Summary
The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. We provide a body of evidence suggesting that SPRTN activates the ATR-CHK1 phosphorylation signalling cascade during physiological DNA replication by proteolysis-dependent eviction of CHK1 from replicative chromatin. The main stimulus for ATR-CHK1 activation is replication protein A (RPA)-coated single-stranded (ss) DNA that typically forms upon DNA replication fork stalling due to uncoupling of the cell division cycle 45 (Cdc45), minichromosome maintenance protein complex 2-7 (Mcm2-7) and go-ichi-ni-san protein complex GINS (CMG) helicase complex from DNA polymerases[5]. This ssDNA-protein structure recruits the ATR–ATR-interacting protein (ATRIP) kinase complex which, with the help of Ewing’s tumour-associated antigen 1 (ETAA1) and DNA topoisomerase.
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