REGULATION OF DNA-REPLICATION ORIGINS DURING CELL-CYCLE PROGRESSION

We have shown previously that chromosome VI of Saccharomyces cerevisia e contains nine origins of DNA replication that differ in initiation f requency and replicate sequentially during the S phase of the cell cyc le(1,2). Here we show that there are links between activation of these multiple origins and regulation of S-phase progression. We study the effects of a DNA-damaging agent, methyl methane sulphonate (MMS), and of mutations in checkpoint genes such as rad53 (ref. 3) on the activit y of origins, measured by two-dimensional gel analysis, and on cell-cy cle progression, measured by fluorescence-activated cell sorting. We f ind that when MMS slows down S-phase progression it also selectively b locks initiation from late origins. A rad53 mutation enhances late and /or inefficient origins and releases the initiation block by MMS. Muta tion of rad53 also results in a late origin becoming early replicating , We conclude that rad53 regulates the timing of initiation of replica tion from late origins during normal cell growth and blocks initiation from late origins in MMS-treated cells. rad53 is, therefore, involved in the cell's surveillance of S-phase progression(4,5). We also find that orc2, which encodes subunit 2 of the origin-recognition complex(6 ,7), is involved in suppression of late origins.