Abstract
Replication origins in eukaryotes form a base for assembly of the pre-replication complex (pre-RC), thereby serving as an initiation site of DNA replication. Characteristics of replication origin vary among species. In fission yeast Schizosaccharomyces pombe, DNA of high AT content is a distinct feature of replication origins; however, it remains to be understood what the general molecular architecture of fission yeast origin is. Here, we performed ChIP-seq mapping of Orc4 and Mcm2, two representative components of the pre-RC, and described the characteristics of their binding sites. The analysis revealed that fission yeast efficient origins are associated with two similar but independent features: a ≥15 bp-long motif with stretches of As and an AT-rich region of a few hundred bp. The A-rich motif was correlated with chromosomal binding of Orc, a DNA-binding component in the pre-RC, whereas the AT-rich region was associated with efficient binding of the DNA replicative helicase Mcm. These two features, in combination with the third feature, a transcription-poor region of approximately 1 kb, enabled to distinguish efficient replication origins from the rest of chromosome arms with high accuracy. This study, hence, provides a model that describes how multiple functional elements specify DNA replication origins in fission yeast genome.
Highlights
DNA replication in eukaryotes initiates bidirectionally at multiple distinct sites in the genome called DNA replication origins
To reveal DNA sequence signatures associated with origin recognition complex (Orc) and Mcm-binding sites in fission yeast replication origins, we determined genome-wide binding locations of pre-RC components, Orc4 and Mcm2, at high resolution by epitope tagbased Chromatin immunoprecipitation (ChIP)-seq
Cdc10-V50 cells expressing PK epitope-tagged Orc4 or Mcm2 were arrested in G1 phase and subjected to anti-PK ChIP-seq analysis
Summary
DNA replication in eukaryotes initiates bidirectionally at multiple distinct sites in the genome called DNA replication origins. A great deal of work over the last few decades has revealed how the pre-replication complex ( pre-RC) is assembled and activated at the origins to ensure concordant initiation of DNA replication during the cell cycle [1,2,3,4]. The bound Orc recruits the DNA replicative helicase Mcm (consisting of the Mcm hexameric complex) onto DNA with the help of the Cdc and Cdt proteins, which are functional only in G1 phase. At the onset of S phase, phosphorylation by Cdk and Ddk kinases triggers recruitment of several additional factors, including DNA polymerases, onto the pre-RC, leading to formation of an active replicative complex and the initiation of replication. Despite a deep understanding of trans-acting factors required for ‘once and only once’ replication initiation during the cell cycle, the nature of the cis-acting elements that define the origins remains to be elucidated in most eukaryotes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
