Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway
AuthorsZaher, Manal S.
Joudeh, Luay I
Sobhy, Mohamed Abdelmaboud
Hingorani, Manju M
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
MetadataShow full item record
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.
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.
SponsorsKing 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.
PublisherOxford University Press (OUP)
JournalNucleic Acids Research
Except where otherwise noted, this item's license is described as This 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 firstname.lastname@example.org
- Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal.
- Authors: Rossi ML, Pike JE, Wang W, Burgers PM, Campbell JL, Bambara RA
- Issue date: 2008 Oct 10
- Dna2p helicase/nuclease is a tracking protein, like FEN1, for flap cleavage during Okazaki fragment maturation.
- Authors: Kao HI, Campbell JL, Bambara RA
- Issue date: 2004 Dec 3
- Significance of the dissociation of Dna2 by flap endonuclease 1 to Okazaki fragment processing in Saccharomyces cerevisiae.
- Authors: Stewart JA, Campbell JL, Bambara RA
- Issue date: 2009 Mar 27
- Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae.
- Authors: Stewart JA, Miller AS, Campbell JL, Bambara RA
- Issue date: 2008 Nov 14
- An alternative pathway for Okazaki fragment processing: resolution of fold-back flaps by Pif1 helicase.
- Authors: Pike JE, Henry RA, Burgers PM, Campbell JL, Bambara RA
- Issue date: 2010 Dec 31