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We investigated the kinetic and substrate-binding characteristics of wild-type and mutant nucleases in relation to the proposed model. Five basic residues R33, K215, K241, R172, and R216, are all implicated in binding branched DNA substrates. All these residues except R172 are involved in binding to duplex DNA carrying a 5\u2032 overhang. Replacement of either K215 or R216 with a neutral amino acid did not alter\n            <jats:italic>k<\/jats:italic>\n            <jats:sub>cat<\/jats:sub>\n            appreciably. However, these mutant nucleases displayed significantly increased values for\n            <jats:italic>K<\/jats:italic>\n            <jats:sub>d<\/jats:sub>\n            and\n            <jats:italic>K<\/jats:italic>\n            <jats:sub>m<\/jats:sub>\n            . A comparison of flap endonuclease binding to pseudoY substrates and duplexes with a single-stranded 5\u2032 overhang suggests a better model for 5\u2032 nuclease-DNA binding. We propose a major revision to the binding model consistent with these biophysical data.\n          <\/jats:p>","DOI":"10.1073\/pnas.082241699","type":"journal-article","created":{"date-parts":[[2002,7,28]],"date-time":"2002-07-28T22:26:28Z","timestamp":1027895188000},"page":"8542-8547","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":23,"title":["Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding"],"prefix":"10.1073","volume":"99","author":[{"given":"Joe J.","family":"Dervan","sequence":"first","affiliation":[{"name":"Division of Genomic Medicine, University of Sheffield Medical School, Krebs Institute, Beech Hill Road, Sheffield S10 2RX, United Kingdom; Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom; Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom; and Celltech Group plc, 216 Bath Road, Slough, Berkshire SL1 4EN, United Kingdom"}]},{"given":"Min","family":"Feng","sequence":"additional","affiliation":[{"name":"Division of Genomic Medicine, University of Sheffield Medical School, Krebs Institute, Beech Hill Road, Sheffield S10 2RX, United Kingdom; Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom; Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom; and Celltech Group plc, 216 Bath Road, Slough, Berkshire SL1 4EN, United Kingdom"}]},{"given":"Dipak","family":"Patel","sequence":"additional","affiliation":[{"name":"Division of Genomic Medicine, University of Sheffield Medical School, Krebs Institute, Beech Hill Road, Sheffield S10 2RX, United Kingdom; Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom; Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom; and Celltech Group plc, 216 Bath Road, Slough, Berkshire SL1 4EN, United Kingdom"}]},{"given":"Jane A.","family":"Grasby","sequence":"additional","affiliation":[{"name":"Division of Genomic Medicine, University of Sheffield Medical School, Krebs Institute, Beech Hill Road, Sheffield S10 2RX, United Kingdom; Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom; Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom; and Celltech Group plc, 216 Bath Road, Slough, Berkshire SL1 4EN, United Kingdom"}]},{"given":"Peter J.","family":"Artymiuk","sequence":"additional","affiliation":[{"name":"Division of Genomic Medicine, University of Sheffield Medical School, Krebs Institute, Beech Hill Road, Sheffield S10 2RX, United Kingdom; Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom; Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom; and Celltech Group plc, 216 Bath Road, Slough, Berkshire SL1 4EN, United Kingdom"}]},{"given":"Thomas A.","family":"Ceska","sequence":"additional","affiliation":[{"name":"Division of Genomic Medicine, University of Sheffield Medical School, Krebs Institute, Beech Hill Road, Sheffield S10 2RX, United Kingdom; Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom; Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom; and Celltech Group plc, 216 Bath Road, Slough, Berkshire SL1 4EN, United Kingdom"}]},{"given":"Jon 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