title: Sequence-selective DNA cleavage by a chimeric metallopeptide.
abstract: A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as a folded domain and thus cleaves selected sequences. The de novo artificial nuclease LnP3W represents the first small, underivatized peptide that is both active as a nuclease and sequence selective.
authors.affiliation: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Roger T
authors.initials: RT
authors.lastname: Kovacic
authors.forename: Joel T
authors.initials: JT
authors.lastname: Welch
authors.forename: Sonya J
authors.initials: SJ
authors.lastname: Franklin
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry; D002563:Cerium / Q000737:chemistry; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D020832:EF Hand Motifs; D004591:Electrophoresis, Polyacrylamide Gel; D005063:Europium / Q000737:chemistry; D019077:Helix-Turn-Helix Motifs; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002147:Calmodulin; D008667:Metalloproteins; D011993:Recombinant Fusion Proteins; D002563:Cerium; D005063:Europium; D004247:DNA