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A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL.

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cam.issuedOnline2018-08-08
dc.contributor.authorAl-Behadili, Ali
dc.contributor.authorUhler, Jay P
dc.contributor.authorBerglund, Anna-Karin
dc.contributor.authorPeter, Bradley
dc.contributor.authorDoimo, Mara
dc.contributor.authorReyes, Aurelio
dc.contributor.authorWanrooij, Sjoerd
dc.contributor.authorZeviani, Massimo
dc.contributor.authorFalkenberg, Maria
dc.contributor.orcidReyes Tellez, Aurelio [0000-0003-2876-2202]
dc.date.accessioned2018-11-14T00:31:39Z
dc.date.available2018-11-14T00:31:39Z
dc.date.issued2018-10-12
dc.description.abstractThe role of Ribonuclease H1 (RNase H1) during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) is a key, yet poorly understood, step in mitochondrial DNA maintenance. Here, we reconstitute the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates. The process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. We find that RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5'-end ribonucleotides disturb ligation, a conclusion which is supported by analysis of RNase H1-deficient patient cells. A second nuclease is therefore required to remove the last ribonucleotides and we demonstrate that Flap endonuclease 1 (FEN1) can execute this function in vitro. Removal of RNA primers at OriL thus depends on a two-nuclease model, which in addition to RNase H1 requires FEN1 or a FEN1-like activity. These findings define the role of RNase H1 at OriL and help to explain the pathogenic consequences of disease causing mutations in RNase H1.
dc.format.mediumPrint
dc.identifier.doi10.17863/CAM.32428
dc.identifier.eissn1362-4962
dc.identifier.issn0305-1048
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/285058
dc.languageeng
dc.language.isoeng
dc.publisherOxford University Press (OUP)
dc.publisher.urlhttp://dx.doi.org/10.1093/nar/gky708
dc.rightsAttribution-NonCommercial 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by-nc/4.0/
dc.subjectDNA Replication
dc.subjectDNA, Mitochondrial
dc.subjectFlap Endonucleases
dc.subjectHumans
dc.subjectMitochondria
dc.subjectMitochondrial Proteins
dc.subjectRNA
dc.subjectRecombinant Proteins
dc.subjectRibonuclease H
dc.subjectRibonucleotides
dc.titleA two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL.
dc.typeArticle
dcterms.dateAccepted2018-07-24
prism.endingPage9483
prism.issueIdentifier18
prism.publicationDate2018
prism.publicationNameNucleic Acids Res
prism.startingPage9471
prism.volume46
pubs.funder-project-idMedical Research Council (MC_UP_1002/1)
pubs.funder-project-idMedical Research Council (MC_EX_MR/P007031/1)
pubs.funder-project-idEuropean Research Council (322424)
pubs.funder-project-idMedical Research Council (MC_UU_00015/8)
pubs.funder-project-idMRC (MC_UU_00015/8)
pubs.funder-project-idMedical Research Council (MC_UU_00015/7)
rioxxterms.licenseref.startdate2018-10
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.typeJournal Article/Review
rioxxterms.versionVoR
rioxxterms.versionofrecord10.1093/nar/gky708

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