T4 endonuclease VII. Importance of a histidine-aspartate cluster within the zinc-binding domain.
The DNA junction-resolving enzyme endonuclease VII of bacteriophage T4 contains a zinc-binding region toward the N-terminal end of the primary sequence. In the center of this 39-amino acid section (between residues 38 and 44) lies the sequence HLDHDHE, termed the His-acid cluster. Closely related sequences are found in three other proteins that have similar zinc-binding motifs. We have analyzed the function of these residues by a site-directed mutagenesis approach, modifying single amino acids and studying the properties of the resulting N-terminal protein A fusions. No sequence changes within the His-acid cluster led to a change in zinc content of the protein, indicating that these residues are not involved in the coordination of zinc. We found that the N-terminal aspartate residue (Asp-40) and the two histidine residues (His-41 and His-43) within the cluster are essential for junction-cleavage activity of the proteins. However, all sequence variations within this region generate proteins that retain their ability to bind to four-way DNA junctions (with minor changes in binding affinity in some cases) and to distort their global structure in the same manner as active enzymes. We conclude that the process of cleavage can be uncoupled from those of binding to and distortion of the junction. It is probable that some amino acid side chains of the His-acid cluster participate in the phosphodiester cleavage mechanism of endonuclease VII. The essential aspartate residue might be required for coordination of catalytic metal ions.
Giraud-Panis
M J
MJ
Cancer Research Campaign Nucleic Acid Structure Research Group, Department of Biochemistry, The University of Dundee, Dundee DD1 4HN, United Kingdom. dmjlilley@bad.dundee.ac.uk
Lilley
D M
DM
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D017122:Bacteriophage T4 / Q000201:enzymology; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015032:Zinc / Q000032:analysis
D004268:DNA-Binding Proteins; D008667:Metalloproteins; D001224:Aspartic Acid; D006639:Histidine; D004247:DNA; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII; D015032:Zinc