Enzymatic formation and resolution of Holliday junctions in vitro

Müller, Berndt; Jones, Christine; Kemper, Börries; West, Stephen C.

doi:10.1016/0092-8674(90)90747-3

Cited by 48 publications

(30 citation statements)

References 34 publications

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“…The strand exchange reaction (100 ill) was performed as described (Miiller et al 1990) using gDNA (12 ~M as nucleotide concentration) and 5'-a~P -labeled linear duplex DNA (10 ~xM), which had been cut with HincII. After incubation for 12 rain at 37°C, the reaction was stopped by adding SDS (0.5%), EDTA {50 raM), and proteinase K (2 mg/ml) and incubated for an additional 30 min at 37°C.…”

Section: Preparation Of Recombination Intermediatesmentioning

confidence: 99%

Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration.

Iwasaki¹,

Takahagi²,

Nakata³

et al. 1992

Genes Dev.

174

131

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The Escherichia coli ruvA and ruvB genes are involved in DNA repair and in the late step of homologous genetic recombination. We have demonstrated previously that the RuvA-RuvB protein complex in the presence of ATP promotes reabsorption of cruciform structures extruded from a supercoiled plasmid with an inverted repeat sequence. Because the cruciform structure is topologically analogous to the Holliday structure, we have proposed that the role of the RuvA and RuvB proteins in recombination is to promote a strand exchange reaction at the Holliday junction. Here, we studied the specific interaction of the RuvA-RuvB complex with the Holliday structure using synthetic analogs prepared by annealing four oligonucleotides. The affinities of the RuvA protein for synthetic Holliday junctions are much higher (>20-fold) than for duplex DNA, and the affinities of the RuvA protein for the junctions are further enhanced (>4-fold) by the interaction with the RuvB protein. The RuvA-RuvB protein complex in the presence of ATP promotes dissociation of the synthetic Holliday junction with homology in the central core into two halves by catalyzing branch migration to the DNA ends, but it does not affect the structure of the synthetic Holliday junction without the homology. The separation of the synthetic Holliday junction is a result of the activity of the RuvA-RuvB complex that promotes strand exchange and DNA unwinding. Furthermore, RuvA and RuvB promote the strand exchange reaction at the Holliday junctions made by RecA. These results provide further evidence that the RuvA-RuvB complex recognizes the Holliday junction and promotes branch migration in homologous recombination.

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Section: Preparation Of Recombination Intermediatesmentioning

confidence: 99%

Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration.

Iwasaki¹,

Takahagi²,

Nakata³

et al. 1992

Genes Dev.

174

131

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“…These substrates include Holliday structures from in vivo-recombined figure-eight plasmid molecules [3], in vitrogenerated RecA-mediated Holliday structures [4], supercoiled and hybrid cruciforms [S], synthetic cruciforms [2,, synthetic Y-structures [2,12,131, heteroduplex loops [14, 151, fivearm and six-arm branched DNAs [16], mismatches in doublestranded DNA [2,14,151, single-strand overhangs [2, 121, single-stranded loops at the ends of hairpins [2], nicks and gaps in dsDNA 12, 121, curved DNA [17], bulky adducts [18, 191 and apurinic sites in dsDNA (Greger. B. and Kemper, B., unpublished results).…”

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confidence: 99%

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

Gölz

Christoph

Birkenkamp‐Demtröder

et al. 1997

European Journal of Biochemistry

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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 ...

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“…In studies reported elsewhere (9), the purified RuvC protein has been shown to cleave Holliday junctions generated in strand exchange reactions catalyzed in vitro by RecA. Two other nucleases with this property have been studied in detail, namely, phage T4 endonuclease VII (14,18) and phage T7 endonuclease I (7,8,19). We found no obvious homology between these proteins and RuvC.…”

mentioning

confidence: 53%

“…An essential intermediate in these reactions is a heteroduplex joint, or Holliday junction, in which the two DNA duplexes are held together by the exchanged strands. Recombinant molecules are produced by specific endonucleolytic cleavage of this intermediate at the point of exchange (18,19). Connolly and West (6) detected a nuclease with this ability in E. coli cell extracts by assaying for specific cleavage of Holliday junctions in an ongoing strand exchange reaction driven by RecA.…”

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confidence: 99%

Resolution of Holliday junctions in Escherichia coli: identification of the ruvC gene product as a 19-kilodalton protein

Sharples

Lloyd

1991

J Bacteriol

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). The gene was located between aspS and the ruvAB operon by DNA sequencing and deletion analysis of ruvC plasmids and was shown to encode a protein of 18,747 Da. Analysis of the DNA flanking ruvC indicated that the gene is transcribed independently of the LexA-regulated ruvAB operon and is not under direct SOS control. ruvC lies downstream of an open reading frame, orf-33, for a protein which migrates during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 33-kDa polypeptide. These two genes probably form an operon. However, expression of ruvC was found to be very poor relative to that of orf-33. A double ribosomal frameshift between these genes is proposed as a possible reason for the low level of RuvC. Two further open reading frames of unknown function were identified, one on either side of the orf-33-ruvC operon.

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Enzymatic formation and resolution of Holliday junctions in vitro

Cited by 48 publications

References 34 publications

Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration.

Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration.

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

Resolution of Holliday junctions in Escherichia coli: identification of the ruvC gene product as a 19-kilodalton protein

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