RESEARCH SUMMARY

We study the regulation of eukaryotic DNA replication. Defects in the replicative machinery often result in DNA damage and/or genome instability. Our approach is to characterize the molecular abnormalities arising in DNA replication caused by mutations in replication regulators naturally associated to tumourigenesis, to unveil new regulatory pathways and to understand how disease is produced.

Cyclin-dependent kinase (CDK) complexes regulate the initiation of DNA replication, activating fork firing at origins of DNA replication at the G1/S transition, and inhibiting origin licensing to ensure a complete, unique replication per cell cycle. Absence of CDK in G1 is essential for optimal origin licensing.CDK upregulation in G1 causes genome instability and is oncogenic.

Recent work by others in yeast and human cells show that CDK upregulation in G1 induces an abnormal, lengthened S phase, with increased DNA damage including double strand breaks. One obvious consequence of increased CDK activity in G1 is reduced origin licensing and firing, as described for a few origins in yeast. What is molecularly different from normal in S phase, how and where damage is produced, and how it induces genome instability, is poorly understood. We approach these questions in Saccharomyces cerevisiae yeast cells, which have proved useful in cell cycle and DNA replication studies.

CDK/cyclin B complexes are maintained inactive in G1, mainly by cyclin degradation of mitotic cyclins by ubiquitination dependent on Cdh1-APC/C (anaphase-promoting complex/cyclosome) and CDK/cyclin inhibition by Sic1 (orthologue to p27 in animal cells), so origin licensing is allowed.

At start, G1 cyclins are synthesised to activate G1-CDK, which in turn activates S-CDK, and inhibits licensing. Sic1 inhibits S CDK activity, necessary for fork firing at G1/S, until Sic1 degradation is promoted by G1-CDK.

Our results with cells lacking Sic1 and/or Cdh1 show, first, that most replication origins studied maintain normal efficiency (resistant origins), and only some (30%) lose efficiency (sensitive origins) to a different extent. Second, origin sensitivity is independent of normal origin firing timing during S phase, origin activity, or origin location on chromosomes, as observed in contiguous origins. Third, cells do not compensate the reduction in forks by firing of silent origins. Fourth, the rate of gross chromosomal rearrangement (GCR) increases close to the sensitive origin ARS507.

All these results suggest that when CDK is deregulated, chromosome regions proximal to sensitive origins are more prone to genome instability. Whether GCR occur by attempts to segregate partially unreplicated chromosomes or by fork progression impediments is unknown.