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) 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.
KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: For financial support, we thank NCI (PO1CA92584 to J.A.T.); BBSRC (grants BB/K009079/1 and BB/M00404X/1 to J.A.G.); the Ministry of Higher Education Libya, EPSRC and the University of Sheffield (studentships to S.I.A., S.J.S. and E.J., respectively); NIH (R01GM060987 to S.M. and R01GM110387 to S.T.); and KAUST for core and CRG3 funding to S.M.H. and J.A.T. J.A.T. acknowledges support by a Robert A. Welch Chemistry Chair, the Cancer Prevention and Research Institute of Texas, and the University of Texas System Science and Technology Acquisition and Retention. The tumour mutation analysis includes data generated by the TCGA Research Network: http://cancergenome.nih.gov/. We thank the ALS, SSRL, the IDAT program, the DOE BER and the NIH project MINOS (R01GM105404) for X-ray data facilities. We thank James Holton for aiding X-ray data analysis.