Eukaryotic genomes often contain more potential replication origins than ar
e actually used during S phase. The molecular mechanisms that prevent some
origins from firing are unknown. Here we show that dormant replication orig
ins on the left arm of budding yeast chromosome III become activated when b
oth passive replication through them is prevented and the Mec1/Rad53 checkp
oint that blocks late-origin firing is inactivated. Under these conditions,
dormant origins fire very late relative to other active origins. These exp
eriments show that some dormant replication origins are competent to fire d
uring S phase and that passage of a replication fork through such origins c
an inactivate them.