T4 endonuclease VII cleaves holliday structures

Mizuuchi, Kiyoshi; Kemper, Börries; Hays, John B.; Weisberg, Robert A.

doi:10.1016/0092-8674(82)90152-0

Cited by 273 publications

(146 citation statements)

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“…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.…”

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confidence: 99%

“…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 B. and Kemper, B., unpublished results).…”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%

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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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confidence: 99%

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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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“…The MMR in T4 is rather a manifestation of the process aimed at the repair of DNA secondary structure. T4 DNA undergoes a complex processing before packaging, which includes resolution or repair of branched and loop-containing structures (66,93) . The T4 marker-dependent recombination must be related to such processing aimed at restoring distorted areas of the double helix.…”

Section: The Biological Signifi Cance Of Mmr In Phage T4mentioning

confidence: 99%

“…The purifi ed enzyme cleaves specifi cally at secondary structures in double-stranded DNA. These structures include branched DNAs, such as HJs (66,67) , Y structures (68) , single-stranded overhangs (69,70) , base mispairings and heteroduplex loops (71 -73) , and bulky adducts (74) . The enzyme does not attack the looping single strand (71) .…”

Section: Enzymes Of Mmr Endonuclease Vii: Recognition and Cuttingmentioning

confidence: 99%

Mismatch repair in recombination of bacteriophage T4

Shcherbakov

2012

BioMolecular Concepts

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The review focuses on the mechanism of mismatch repair in bacteriophage T4. It was fi rst observed in T4 as an extra recombination mechanism, which contributed to the general recombination only when particular rII mutations were used as genetic markers (high-recombination markers), whereas it was inactive toward other rII mutations (lowrecombination markers). This marker-dependent recombination pathway was identifi ed as a repair of mismatches in recombinational heteroduplexes. Comparison of the structure of markers enabled us to make several specifi c conclusions on the nature of the marker discrimination by the mismatch repair system operating during T4 crosses. First, heteroduplexes with one mismatched base pair (either of transition or of transversion type) as well as single-nucleotide mismatches of indel type are not effi ciently repaired. Second, among the repairable mismatches, those with two or more contiguous mismatched nucleotides are the most effectively repaired, whereas insertion of one correct pair between two mismatched ones reduces the repairability. Third, heteroduplexes containing insertion mutations are repaired asymmetrically, the longer strand being preferentially removed. Fourth, the sequence environment is an important factor. Inspection of the sequences fl anking mismatches shows that runs of A:T pairs directly neighboring the mismatches greatly promote repair. The mismatch is recognized by T4 endonuclease VII and nicked on the 3 ′ side. The nonpaired 3 ′ terminus is attacked by the proofreading 3 ′ → 5 ′ exonuclease of T4 DNA polymerase that removes the mismatched nucleotides along with several ( ∼ 25) complementary nucleotides (the repair tract) and then switches to polymerization. The residual nick is ligated by DNA ligase (gp30). Most probably, the T4 system repairs replication and other mismatches as well; however, it might not discriminate old and new DNA strands and so does not seem to be aimed at repair of replication errors, in contrast to the most commonly studied examples of mismatch repair.

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“…The T4 Holliday junction-cleaving enzyme (EndoVII), the product of gene 49, is a structure-specific endonuclease and was the first enzyme shown to resolve Holliday junctions (12). EndoVII-deficient infections accumulate an abundance of heavily branched intermediates (13).…”

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Regression supports two mechanisms of fork processing in phage T4

Long

Kreuzer

2008

Proc. Natl. Acad. Sci. U.S.A.

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Replication forks routinely encounter damaged DNA and tightly bound proteins, leading to fork stalling and inactivation. To complete DNA synthesis, it is necessary to remove fork-blocking lesions and reactivate stalled fork structures, which can occur by multiple mechanisms. To study the mechanisms of stalled fork reactivation, we used a model fork intermediate, the origin fork, which is formed during replication from the bacteriophage T4 origin, ori(34). The origin fork accumulates within the T4 chromosome in a site-specific manner without the need for replication inhibitors or DNA damage. We report here that the origin fork is processed in vivo to generate a regressed fork structure. Furthermore, origin fork regression supports two mechanisms of fork resolution that can potentially lead to fork reactivation. Fork regression generates both a site-specific double-stranded end (DSE) and a Holliday junction. Each of these DNA elements serves as a target for processing by the T4 ATPase/exonuclease complex [gene product (gp) 46/47] and Holliday junction-cleaving enzyme (EndoVII), respectively. In the absence of both gp46 and EndoVII, regressed origin forks are stabilized and persist throughout infection. In the presence of EndoVII, but not gp46, there is significantly less regressed origin fork accumulation apparently due to cleavage of the regressed fork Holliday junction. In the presence of gp46, but not EndoVII, regressed origin fork DSEs are processed by degradation of the DSE and a pathway that includes recombination proteins. Although both mechanisms can occur independently, they may normally function together as a single fork reactivation pathway.2D gel electrophoresis ͉ DNA replication ͉ recombination ͉ restart R eplication forks routinely encounter various lesions, ranging from damaged nucleotide residues to covalent protein-DNA complexes. The consequences of these encounters depend on the type and position of the lesion (1). Although some lesions result in fork breakage, many result in fork stalling and replisome inactivation. Presumably, replisome disassembly is important for the repair of the original DNA lesion. However, it is then necessary to reactivate stalled forks to complete replication and maintain genome stability. Therefore, it is critical to understand the mechanisms of stalled fork resolution and reactivation.To identify the pathways of stalled fork reactivation, we have used a model fork intermediate that is formed during replication from bacteriophage T4 origin, ori(34). Replication from ori(34) occurs bidirectionally through a two-step process (Fig. 1) (2). A middle-mode promoter and DNA unwinding element promote the formation of an R loop that initiates the first replication fork (3). The mechanism of T4 replication from an R loop has been analyzed in vitro (4). Initially, the branch structure of the R loop supports the binding of the replicative helicase loader [gene product (gp) 59], which promotes removal of the ssDNA-binding protein (gp32) in favor of the replicative helicase (gp41). ...

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T4 endonuclease VII cleaves holliday structures

Cited by 273 publications

References 26 publications

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

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

Mismatch repair in recombination of bacteriophage T4

Regression supports two mechanisms of fork processing in phage T4

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