Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway.
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Authors
Zaher, Manal S
Rashid, Fahad
Song, Bo
Sobhy, Mohamed A
Tehseen, Muhammad
Hingorani, Manju M
Hamdan, Samir M
Publication Date
2018-04-06Journal Title
Nucleic Acids Res
ISSN
0305-1048
Publisher
Oxford University Press (OUP)
Volume
46
Issue
6
Pages
2956-2974
Language
eng
Type
Article
Physical Medium
Print
Metadata
Show full item recordCitation
Zaher, M. S., Rashid, F., Song, B., Joudeh, L., Sobhy, M. A., Tehseen, M., Hingorani, M. M., & et al. (2018). Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway.. Nucleic Acids Res, 46 (6), 2956-2974. https://doi.org/10.1093/nar/gky082
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.
Keywords
Saccharomyces cerevisiae, DNA Helicases, Acetyltransferases, Saccharomyces cerevisiae Proteins, Membrane Proteins, DNA, DNA, Fungal, Fluorescence Resonance Energy Transfer, Signal Transduction, DNA Replication, Protein Binding, Substrate Specificity, Kinetics, Replication Protein A, DNA Cleavage, Single Molecule Imaging
Identifiers
External DOI: https://doi.org/10.1093/nar/gky082
This record's URL: https://www.repository.cam.ac.uk/handle/1810/280200
Rights
Attribution-NonCommercial 4.0 International
Licence URL: https://creativecommons.org/licenses/by-nc/4.0/
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