The modular character of a DNA junction-resolving enzyme: a zinc-binding motif in bacteriophage T4 endonuclease VII.
Bacteriophage T4 endonuclease VII is one of a class of structure-selective enzymes that resolve helical branchpoints in DNA molecules. The sequence of this protein suggests a modular organisation. We have expressed a synthetic gene encoding endonuclease VII, which has been used in a directed mutagenesis exercise, with the aim of understanding the role of different sections of the protein sequence. Towards the N-terminal end of the protein lies a section of polypeptide in which four cysteine residues distributed in a CxxC--CxxC pattern co-ordinate one atom of zinc. The N-terminal section composed of amino acid residues 1 to 65 isolated from the remaining C-terminal section also binds one mole of zinc, suggesting that this region folds autonomously. Mutation shows that the outer cysteine residues are essential for zinc binding, while the inner cysteine residues are partially degenerate in that either one of the two (but not both) can be replaced while retaining some zinc. The activity as a junction-resolving enzyme correlated qualitatively with the presence of the zinc. In the C-terminal part of the protein lies a section that is 48% identical with a sequence found in the DNA repair protein T4 endonuclease V. We can replace the section of T4 endonuclease VII with the corresponding sequence from T4 endonuclease V with no change in the pattern of cleavage on four-way junctions. The evidence supports a modular construction for T4 endonuclease VII.
Giraud-Panis
M J
MJ
Department of Biochemistry, The University, Dundee, UK.
Duckett
D R
DR
Lilley
D M
DM
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005813:Genes, Synthetic; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D006639:Histidine; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; C035571:endodeoxyribonuclease VII; D043224:Deoxyribonuclease (Pyrimidine Dimer); D015032:Zinc; D003545:Cysteine