Checkpoint kinase 1 maintains genomic stability independently of its contribution to DNA replication

MARINA A. GONZÁLEZ BESTEIRO; NICOLÁS L. CALZETTA; M. BELÉN DE LA VEGA; NATALIA PAVIOLO; MARÍA BELÉN FEDERICO; SABRINA FLORENCIA MANSILLA; AGOSTINA BERTOLÍN; VANESA GOTTIFREDI

Trieste

Congreso; At the intersection of DNA replication and Genome Maintenance: From Mechanisms to Therapy; 2016

ICGEB

The encounter of replication forks with DNA lesions activate Checkpoint Kinase 1 (Chk1), which stabilizes forks and inhibits replication origin firing. While the mechanisms that underlie Chk1-dependent inhibition of origin firing have been elucidated, the downstream effectors of Chk1 that control replication fork progression remain largely unexplored. We observed that roscovitine, a CDK (cyclin-dependent kinase) inhibitor, alleviates the exacerbated origin firing and impaired fork progression phenotypes caused by Chk1 depletion. Others have proposed that roscovitine restores the progression of active forks by reducing origin firing, thereby making the nucleotide pool available for DNA elongation. However, we provide evidence that this is not the case, since the reduction in origin firing back to wild-type levels in Chk1-depleted cells (by means other than roscovitine) does not translate into normal DNA elongation rates. Conversely, we propose that DNA lesions created by Chk1 loss account for slow replication fork progression.These results prompted us to test whether the genomic instability observed in Chk1-depleted cells results from increased origin firing or reduced fork rates. Interestingly, while exacerbated origin firing promotes H2AX accumulation in Chk1-inhibited cells, impaired replication fork elongation does not. Surprisingly however, MUS81-mediated double-strand break (DSB) formation in Chk1-inhibited cells is not rescued by restoring either origin firing or fork elongation. In agreement, MUS81-dependent genomic instability of Chk1-depleted cells arises independently of the status of such replication parameters. Moreover, although MUS81 depletion rescues DSB formation and genomic instability of Chk1-depleted U2OS cells, origin firing and fork elongation phenotypes in Chk1-depleted and Chk1- and MUS81-depleted cells are similar.Our results thus imply that the profoundly altered DNA replication program of Chk1-depleted cells is not a determinant of MUS81-mediated DSB formation and genomic instability. Similarly, DSB accumulation in Chk1-depleted cells does not impact on the DNA replication program. In conclusion, our study suggests that Chk1 regulates replication fork progression, origin firing and DSB-triggered genomic stability by independent pathways.