Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

Handle URI:
http://hdl.handle.net/10754/627120
Title:
Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway
Authors:
Zaher, Manal S.; Rashid, Fahad; Song, Bo; Joudeh, Luay I; Sobhy, Mohamed Abdelmaboud; Tehseen, Muhammad ( 0000-0001-9834-2372 ) ; Hingorani, Manju M; Hamdan, Samir ( 0000-0001-5192-1852 )
Abstract:
RNA-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.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program
Citation:
Zaher MS, Rashid F, Song B, Joudeh LI, Sobhy MA, et al. (2018) Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway. Nucleic Acids Research. Available: http://dx.doi.org/10.1093/nar/gky082.
Publisher:
Oxford University Press (OUP)
Journal:
Nucleic Acids Research
Issue Date:
27-Jan-2018
DOI:
10.1093/nar/gky082
Type:
Article
ISSN:
0305-1048; 1362-4962
Sponsors:
King Abdullah University of Science and Technology Core Funding (to S.M.H.); Competitive Research Award (CRG3) (to S.M.H.); National Institutes of Health [R15 GM114743 to M.M.H.]. Funding for open access charge: Global Collaborative Research, King Abdullah University of Science and Technology.
Additional Links:
https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gky082/4840237
Appears in Collections:
Articles; Bioscience Program; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorZaher, Manal S.en
dc.contributor.authorRashid, Fahaden
dc.contributor.authorSong, Boen
dc.contributor.authorJoudeh, Luay Ien
dc.contributor.authorSobhy, Mohamed Abdelmabouden
dc.contributor.authorTehseen, Muhammaden
dc.contributor.authorHingorani, Manju Men
dc.contributor.authorHamdan, Samiren
dc.date.accessioned2018-02-13T13:43:18Z-
dc.date.available2018-02-13T13:43:18Z-
dc.date.issued2018-01-27en
dc.identifier.citationZaher MS, Rashid F, Song B, Joudeh LI, Sobhy MA, et al. (2018) Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway. Nucleic Acids Research. Available: http://dx.doi.org/10.1093/nar/gky082.en
dc.identifier.issn0305-1048en
dc.identifier.issn1362-4962en
dc.identifier.doi10.1093/nar/gky082en
dc.identifier.urihttp://hdl.handle.net/10754/627120-
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.en
dc.description.sponsorshipKing Abdullah University of Science and Technology Core Funding (to S.M.H.); Competitive Research Award (CRG3) (to S.M.H.); National Institutes of Health [R15 GM114743 to M.M.H.]. Funding for open access charge: Global Collaborative Research, King Abdullah University of Science and Technology.en
dc.publisherOxford University Press (OUP)en
dc.relation.urlhttps://academic.oup.com/nar/advance-article/doi/10.1093/nar/gky082/4840237en
dc.rightsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.comen
dc.rights.urihttp://creativecommons.org/licenses/by-nc/4.0/en
dc.titleMissed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathwayen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.contributor.departmentBioscience Programen
dc.identifier.journalNucleic Acids Researchen
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionDepartment of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA.en
kaust.authorZaher, Manal S.en
kaust.authorRashid, Fahaden
kaust.authorJoudeh, Luay Ien
kaust.authorSobhy, Mohamed Abdelmabouden
kaust.authorTehseen, Muhammaden
kaust.authorHamdan, Samiren
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