Our results reveal that at least one-third of replication origins in yeast genome are transcriptionally active, despite their intergenic location. We also confirm that RNAPII-dependent transcription inhibits pre-RC formation and thus abolishes replication origin firing. Nevertheless, our results show that transcription-coupled inactivation of replication origins is reversible in G1, and pre-RC components can be quickly reloaded to origins when transcription stops. Relicensing of transcriptionally inactivated origins fully restores their functionality and leads to their firing in S phase.
Several recent studies suggest that active transcription abolishes replication origin firing by inhibiting pre-RC formation (1
). Although the majority of replication origins in budding yeast are located in intergenic regions of the genome, recent data show that transcription of noncoding regions is widespread in yeast (6
). To estimate the fraction of annotated replication origins that are regularly transcribed, we reanalyzed the genome-wide data of yeast transcripts (7
) in the loci of replication origins (12
). Our analysis shows that at least one-third of the replication origin sequences are transcribed on regular basis, mostly as CUTs (). In addition, several replication origins are located in otherwise transcriptionally active regions that have significant transcriptional overlap due to normal gene expression (). However, the replication origin placement in yeast genome seems to favor transcriptionally more silent regions, as the number of heavily transcribed origins is significantly smaller than the ones transcribed occasionally (). Although direct transcription through the origins disrupts their function, the replication machinery might benefit from transcription-coupled remodeling and modifications of the chromatin that might help pre-RC components to access DNA and facilitate the initiation of replication. The fact that many replication origins are transcribed raises question about the fate of transcribed origins. As there is an excess of potential origins on chromosomes, cells can afford the inactivation of some of them under normal circumstances. Indeed, not all origins fire in a single S phase, and even the most efficient origins do not fire in every S phase (24
). However, the activity of those dormant origins becomes critical in stress conditions, when the movement of replication forks initiated from major origins stalls or slows down (25
). An efficient alternative to the transcription-coupled loss of functional origins would be reestablishing of pre-RCs on origins after transcription. To investigate this possibility, we inserted different replication origins into the coding region of an inducible gene and determined the dynamics of pre-RC components in this locus. Our results show that both ORC and MCM complexes were quickly reloaded to origins as soon as transcription was shut down ( and ), indicating the possible rescue of replication origins by this mechanism. Importantly, in G1
-arrested cells pre-RCs can reform on origins without transition through S phase. After release from G1
arrest, DNA replication initiation protein Cdc45 was recruited to relicensed origin (A
), and DNA replication was initiated in S phase (, C–E
). Based on these results, we propose that pre-RC assembly in yeast is widely perturbed by sporadic transcription; however, when the transcription bubble has passed the loci of replication origins, pre-RCs can quickly reassemble, ensuring that sufficient amounts of functional replication initiation loci will be available to carry out S phase.