Identification of Amino Acids of Endonuclease VII Essential for Binding and Cleavage of Cruciform DNA

Abstract

Endonuclease VII is a Holliday-structure-resolving enzyme of bacteriophage T4. The active protein is a homodimer with 157 amino acidshonomer. An amber mutation (amE727 in codon 151) inactivates the nuclease completely, indicating the importance of the seven C-terminal amino acids for nucleolytic activity. The influence of these amino acids on cruciform-DNA binding and cleavage was investigated through functional analysis of C-terminal-truncated proteins derived from deletion constructs. It was found that the three C-terminal amino acids are not necessary for binding and cleavage. A transition from active to inactive protein occurs gradually with truncations of the next four amino acids. Reduction of DNA-binding ability, as measured by electrophoretic mobility shift assays, was determined to be the primary defect in the cleavage-deficient proteins. This was further concluded by the finding that EVII-(1 -150)-peptide""'h'r, a protein with fairly low affinity to cruciform DNA, contributes cleavage activity to reactions of wild-type EVII with cruciform DNA.[Asp62]EVII-( 1 -156)-peptide lacking one C-termnal amino acid, contains a point mutation in codon 62 that eliminates the nucleolytic activity of the protein while retaining its DNA-binding proficiency. By mixing binding-deficient and cleavage-deficient mutants in the same assay, cleavage of cruciform DNA resumed. Evidence is presented that complementation occurs by heterodimer formation. Our results show that the zinc-binding motif of EVII is not sufficient for cruciform-DNA binding.Keywords: endonuclease VII; DNA-binding protein; bacteriophage T4; DNA repair; recombination.Endonuclease VII (EVII) is the product of gene 49 of phage T4. The endonucleolytic activity of EVII is involved in DNA packaging and mainly required late during the infection cycle [I].Detailed investigations of the substrate specificity of EVII revealed that the enzyme can react with many substrates by introducing staggered nicks flanking the defect within 2-6 nucleotides 11, 21. These substrates include Holliday structures from in vivo-recombined figure-eight plasmid molecules [3] The broad specificity of EVII for structural deviations in dsDNA raised the question of how the enzyme recognizes its target sites. From several studies addressing protein-DNA interactions it become obvious that binding affinities are primarily brought about by electrostatic interactions between basic amino acid residues and the DNA-phosphate backbone. Specifity of binding is accomplished by coordinated placement of basic amino acids, which in turn is determined by the three-dimensional structure of the enzyme. Particular helical segments appear to be widespread among such structural elements [22].Wild-type EVII binds strongly to cruciform DNA, protecting about five residues at the junction point in opposite strands from hydroxyl radical cleavage [7]. As shown here, the protein derived from a classical amber mutation in codon 151 of gene 49 (amE727 [23]) binds very weakly to cruciform DNA. The stop codon truncates ...

Holliday Junction Resolvases

“…T4 endo VII is a dimeric protein that binds preferentially to four-way DNA junctions in a Mg 2þ -and sequence-independent manner (Nishimoto et al 1979;Picksley et al 1990;Giraud-Panis and Lilley 1996;Pöhler et al 1996;Golz et al 1997). Interactions between the amino and carboxyl termini of different monomers are essential for DNA binding (Birkenbihl and Kemper 1998).…”

Structural Maintenance of Chromosomes Protein C-terminal Domains Bind Preferentially to DNA with Secondary Structure

“…21) or CFM13 (28 nucleotides/oligonucleotide; Ref. 22), a 600-bp centromeric region DNA from CEN3 of S. cerevisiae (23); and a 189-bp bent fragment from the Drosophila ftz scaffold-associated region (SAR) with an A/T content of 80% (24), full-length ss or ds M13mp18 and ⌽X174 DNA, and pBR322, pUC19 plasmid DNAs.…”

Inhibition of holliday structure resolving endonuclease VII of bacteriophage T4 by recombination enzymes UvsX and UvsY

“…A likely possibility is that UvsY induces a conformational change of Endo VII, thereby modulating the enzyme activity. Since we have recently shown that binding and cleavage reside in distinct functional domains of Endo VII (Golz et al, 1997) further studies are necessary to determine whether a modulation of Endo VII affects the DNA binding or the nucleolytic activity or both.…”