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dc.contributor.authorZaher, Manal S
dc.contributor.authorRashid, Fahad
dc.contributor.authorSong, Bo
dc.contributor.authorJoudeh, Luay
dc.contributor.authorSobhy, Mohamed A
dc.contributor.authorTehseen, Muhammad
dc.contributor.authorHingorani, Manju M
dc.contributor.authorHamdan, Samir M
dc.date.accessioned2018-09-11T17:32:27Z
dc.date.available2018-09-11T17:32:27Z
dc.date.issued2018-04-06
dc.identifier.issn0305-1048
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/280200
dc.description.abstractRNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.
dc.format.mediumPrint
dc.languageeng
dc.publisherOxford University Press (OUP)
dc.rightsAttribution-NonCommercial 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by-nc/4.0/
dc.subjectSaccharomyces cerevisiae
dc.subjectDNA Helicases
dc.subjectAcetyltransferases
dc.subjectSaccharomyces cerevisiae Proteins
dc.subjectMembrane Proteins
dc.subjectDNA
dc.subjectDNA, Fungal
dc.subjectFluorescence Resonance Energy Transfer
dc.subjectSignal Transduction
dc.subjectDNA Replication
dc.subjectProtein Binding
dc.subjectSubstrate Specificity
dc.subjectKinetics
dc.subjectReplication Protein A
dc.subjectDNA Cleavage
dc.subjectSingle Molecule Imaging
dc.titleMissed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway.
dc.typeArticle
prism.endingPage2974
prism.issueIdentifier6
prism.publicationDate2018
prism.publicationNameNucleic Acids Res
prism.startingPage2956
prism.volume46
dc.identifier.doi10.17863/CAM.27567
dcterms.dateAccepted2018-01-27
rioxxterms.versionofrecord10.1093/nar/gky082
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.licenseref.startdate2018-04
dc.contributor.orcidJoudeh, Luay [0000-0001-9338-205X]
dc.identifier.eissn1362-4962
rioxxterms.typeJournal Article/Review
cam.issuedOnline2018-02-06


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Except where otherwise noted, this item's licence is described as Attribution-NonCommercial 4.0 International