Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Centre for Chemical Biology, Sheffield Institute for Nucleic Acids (SInFoNiA), Department of Chemistry, University of Sheffield, Sheffield S3 7HF, UK.
Nat Commun. 2017 Jun 27;8:15855. doi: 10.1038/ncomms15855.
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.
DNA 复制和修复酶 Flap Endonuclease 1（FEN1）对于基因组完整性至关重要，并且 FEN1 突变出现在多种癌症中。FEN1 精确地切割单链（ss）5'-flap 为双链（ds）DNA。然而，FEN1 如何选择但不切割 ss 5'-flap 仍然是一个谜。在这里，我们结合晶体学、生化和遗传分析表明，两个 dsDNA 结合位点确定了 5'极性，并揭示了静电相互作用对 DNA 磷酸二酯骨架的意外控制。通过“磷酸导向”，碱性残基通过 FEN1 的活性位点中的一个入口在能量上引导反向 ss 5'-flap，并为催化作用移动 dsDNA。这些残基的突变导致体外催化速率降低 18000 倍，体内大规模三核苷酸（GAA）重复扩展，这意味着磷酸导向的失败促进了复制过程中意想不到的滞后链模板转换机制。因此，磷酸导向是 FEN1 的一个未被认识到的功能，它加强了 5'-flap 的特异性和催化作用，防止了基因组不稳定性。
