Human Flap Endonuclease-1:  Conformational Change upon Binding to the Flap DNA Substrate and Location of the Mg2+ Binding Site

• Published in: Biochemistry (Easton) 2001-03, Vol.40 (10), p.3208-3214
• Main Authors: Kim, Chang-Yub, Park, Min S, Dyer, R. Brian
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Amino Acid Substitution - genetics; Aspartic Acid - genetics; Binding Sites - genetics; DNA - metabolism; DNA Repair; DNA Replication; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Flap Endonucleases; Humans; Magnesium - metabolism; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides - metabolism; Protein Binding - genetics; Protein Conformation; Protein Structure, Secondary; Spectroscopy, Fourier Transform Infrared; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi002100n

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is a key enzyme in DNA replication and repair. FEN-1 recognizes the 5‘-flap DNA structure and cleaves it, a specialized endonuclease function essential for the processing of Okazaki fragments during DNA replication and for the repair of 5‘-end single-stranded tails from nicked double-stranded DNA substrates. Magnesium is a cofactor required for nuclease activity. We have used Fourier transform infrared (FTIR) spectroscopy to better understand how Mg2+ and flap DNA interact with human FEN-1. FTIR spectroscopy provides three fundamentally new insights into the structural changes induced by the interaction of FEN-1 with substrate DNA and Mg2+. First, FTIR difference spectra in the amide I vibrational band (1600−1700 cm-1) reveal a change in the secondary structure of FEN-1 induced by substrate DNA binding. Quantitative analysis of the FTIR spectra indicates a 4% increase in helicity upon DNA binding or about 14 residues converted from disordered to helical conformations. The observation that the residues are disordered without DNA strongly implicates the flexible loop region. The conversion to helix also suggests a mechanism for locking the flexible loop region around the bound DNA. This is the first direct experimental evidence for a binding mechanism that involves a secondary structural change of the protein. Second, in contrast with DNA binding, no change is observed in the secondary structure of FEN-1 upon Mg2+ binding to the wild type or to the noncleaving D181A mutant. Third, the FTIR results provide direct evidence (via the carboxylate ligand band at 1535 cm-1) that not only is D181 a ligand to Mg2+ in the human enzyme but Mg2+ binding does not occur in the D181A mutant which lacks this ligand.