Relicensing of Transcriptionally Inactivated Replication Origins in Budding Yeast

Abstract

DNA replication origins are licensed in early G1 phase of the cell cycle where the origin recognition complex (ORC) recruits the minichromosome maintenance (MCM) helicase to origins. These pre-replicative complexes (pre-RCs) remain inactive until replication is initiated in the S phase. However, transcriptional activity in the regions of origins can eliminate their functionality by displacing the components of pre-RC from DNA. We analyzed genome-wide data of mRNA and cryptic unstable transcripts in the context of locations of replication origins in yeast genome and found that at least one-third of the origins are transcribed and therefore might be inactivated by transcription. When investigating the fate of transcriptionally inactivated origins, we found that replication origins were repetitively licensed in G1 to reestablish their functionality after transcription. We propose that reloading of pre-RC components in G1 might be utilized for the maintenance of sufficient number of competent origins for efficient initiation of DNA replication in S phase.

Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA

“…It was previously shown that transcription through an origin can disrupt pre-RCs and inhibit origin function (Looke et al, 2010; Mori and Shirahige, 2007; Snyder et al, 1988; Tanaka et al, 1994). However, although Mcm2-7 complexes are lost from the original loading site after transcription through the origin, we find here that they can be functionally redistributed to downstream sites by elongating RNA polymerase.…”

“…Principally, eukaryotic origin specification mechanisms either actively recruit initiation factors to direct pre-RC assembly at specific chromosomal regions, or conversely exclude pre-RCs from potential origin sites. Transcription, for example, which is known to interfere with pre-RC assembly and thereby inhibit replication origin activity (Donato et al, 2006; Looke et al, 2010; Mori and Shirahige, 2007; Nieduszynski et al, 2005; Snyder et al, 1988), may shape chromosomal replication profiles by restricting pre-RC assembly to non-transcribed regions. Consistent with this notion, onset of zygotic transcription at the midblastula transition promotes specific origin usage in Xenopus and Drosophila embryos by confining replication initiation sites to intergenic regions (Hyrien et al, 1995; Sasaki et al, 1999).…”

“…We reasoned that the ability to slide may help maintain Mcm2-7 DHs on DNA after collision with other proteins. For example, transcriptional activity has been detected at a large fraction of budding yeast origins (Looke et al, 2010), indicating that collisions between RNA polymerase and pre-RCs may occur on a frequent basis. We, therefore, set out to investigate Mcm2-7 DH stability after collision with other proteins on DNA.…”

Large transcription units unify copy number variants and common fragile sites arising under replication stress

“…Importantly, pre-replication complexes (pre-RCs) are only licensed in G1 and must remain bound for firing to occur in S-phase. Movement of RNA polymerase through an origin can sweep away a pre-RC, as demonstrated in yeast (Snyder et al 1988;Looke et al 2010). We thus suggest that dormant origins do not rescue TrDoFFs at CNV hotspots/ CFSs because expansive locus transcription has persisted into S and removed those pre-RCs before they can be utilized (Fig.…”

Fragile sites in cancer: more than meets the eye

“…Importantly, pre-replication complexes (pre-RCs) are only licensed in G1 and must remain bound for firing to occur in S-phase. Movement of RNA pol II through an origin can displace a pre-RC, as demonstrated in yeast 88, 89 . Thus, Wilson et al .…”