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

MARINA A. GONZÁLEZ BESTEIRO, NICOLÁS CALZETTA, M. BELÉN DE LA VEGA, NATALIA PAVIOLO, M. BELÉN FEDERICO, SABRINA F. MANSILLA, AGUSTINA BERTOLÍN AND VANESA GOTTIFREDI.

Trieste

Conferencia; At the intersection of DNA replication and Genome maintenance: from mechanism to teraphy; 2016

ICGEB

The encounter of replication forks with DNA lesions activate Checkpoint Kinase 1 (Chk1), which stabilizes forks and inhibits replication origin firing. The altered DNA replication program in Chk1-depleted cells, evidenced by increased origin firing and slow replication rates, might account for the genomic instability of these cells. However, definite evidence of a link between aberrant replication and loss of genomic stability in Chk1-deficient cells is still lacking. 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. 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 gammaH2AX accumulation in Chk1-inhibited cells, impaired replication fork elongation does not. Surprisingly however, increased double-strand break (DSB) formation in Chk1-inhibited cells is not rescued by restoring origin firing. In agreement, MUS81-dependent genomic instability in Chk1-depleted cells arises independently of the levels of replication origin firing. In conclusion, our results suggest that the profoundly altered DNA replication program of Chk1-depleted cells is not a determinant of MUS81-mediated DSB formation and genomic instability.