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Nat Commun. 2017 Jun 27;8:15855. doi: 10.1038/ncomms15855.

Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability.

Author information

1
Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
2
Centre for Chemical Biology, Sheffield Institute for Nucleic Acids (SInFoNiA), Department of Chemistry, University of Sheffield, Sheffield S3 7HF, UK.
3
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
4
Department of Biology, Tufts University, Medford, Massachusetts 02155, USA.
5
Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
6
Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia.
7
Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

Abstract

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via 'phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability.

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