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dc.contributor.authorSong, Bo
dc.contributor.authorHamdan, Samir
dc.contributor.authorHingorani, Manju M
dc.date.accessioned2018-02-13T13:43:19Z
dc.date.available2018-02-13T13:43:19Z
dc.date.issued2018-02-09
dc.identifier.citationSong B, Hamdan SM, Hingorani MM (2018) Positioning the 5’-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage. Journal of Biological Chemistry: jbc.RA117.001137. Available: http://dx.doi.org/10.1074/jbc.ra117.001137.
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.pmid29462789
dc.identifier.doi10.1074/jbc.ra117.001137
dc.identifier.urihttp://hdl.handle.net/10754/627124
dc.description.abstractFlap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.
dc.description.sponsorshipThe authors would like to thank David Wilson for the hFEN1 clone and Manal Zaher for thoughtful discussions on interpreting the data. This work was supported by NIH grant R15 GM114743 to M.M.H. and a Competitive Research Award (CRG3) by King Abdullah University of Science and Technology to S.M.H. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
dc.publisherAmerican Society for Biochemistry & Molecular Biology (ASBMB)
dc.relation.urlhttp://www.jbc.org/content/early/2018/02/09/jbc.RA117.001137
dc.rightsArchived with thanks to Journal of Biological Chemistry. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
dc.rights.urihttp://www.jbc.org/site/misc/edpolicy.xhtml#copyright
dc.titlePositioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage
dc.typeArticle
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Division
dc.contributor.departmentBioscience Program
dc.identifier.journalJournal of Biological Chemistry
dc.eprint.versionPublisher's Version/PDF
dc.contributor.institutionWesleyan University, United States
dc.contributor.institutionMolecular Biology and Biochemistry, Wesleyan University, United States
kaust.personHamdan, Samir


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