Abstract
Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation.
Highlights
We used various experimental approaches to determine whether Origin G-rich Repeated Element (OGRE)/G4 is a functional element at metazoan origins
Origins were identified from which cells by purification of Short RNA-primed Nascent Strands (SNS), a procedure that we and others repeatedly found to be accurate for origin analysis in Drosophila melanogaster[5], mouse[2,3,4] Arabidopsis thaliana[11], Caenorhabditis elegans[12], chicken[7], and human cells[13,14,15], and the results of which were confirmed by different approaches[6,10,14,15]
We tested the capacity of G4 formation by sequences found in the origin vicinity using isothermal difference spectra (IDS) and circular dichroism (CD)
Summary
We used various experimental approaches to determine whether OGRE/G4 is a functional element at metazoan origins. Using an in vivo genetic approach at an endogenous locus, we showed that deletion of this motif strongly reduced origin activity in mouse cells. An OGRE/G4-containig sequence introduced in an ectopic origin-free region promoted the establishment of a new functional origin. We showed that a plasmid containing an origin with an OGRE/G4 element can replicate in HEK293 cells that express EBNA1 almost as efficiently as plasmids containing the Epstein-Barr virus (EBV) origin OriP, and that deletion of the OGRE/G4 element strongly reduces its replication efficiency. All our results converge to the conclusion that G-rich elements, including the OGRE/G4 motif, are functionally important for origin activity
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