Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

Ichiyanagi, Kenji; Iwasaki, Hiroshi; Hishida, Takashi; Shinagawa, Hideo

doi:10.1046/j.1365-2443.1998.00213.x

Cited by 19 publications

(26 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All RuvC mutants formed a complex with HJ2 DNA as efficiently as wild-type protein in the absence of NaCl. This result is consistent with the previous result (23). In contrast, F69A and F69L did not bind junction DNA in the presence of 100 mM NaCl (Fig.…”

Section: Dna Repair Activities Of Phe-69 Mutants Of Ruvc-ruvc Mutantssupporting

confidence: 93%

“…ruvC gene mutations were confirmed by sequencing. The F69L mutant had been isolated in a previous study (23). Each mutant ruvC gene was carried in pET-8c with the same construction as pRC100.…”

Section: Methodsmentioning

confidence: 99%

“…A previously isolated F69L mutant of RuvC showed no detectable cleavage activity, but formed a dimer that bound a Holliday junction in the same manner as wild-type RuvC, as judged by a gel shift assay in which no salt was added to the reaction mixtures (23). This result suggests that Phe-69 does not directly participate in DNA binding activity detectable by a salt-free gel shift assay, but that it must play an important role in RuvC function.…”

mentioning

confidence: 85%

“…This result suggests that Phe-69 does not directly participate in DNA binding activity detectable by a salt-free gel shift assay, but that it must play an important role in RuvC function. We speculated that this residue makes a stacking interaction with a nucleotide base near the cleavage site (20,23).…”

mentioning

confidence: 99%

See 3 more Smart Citations

Evidence that Phenylalanine 69 in Escherichia coliRuvC Resolvase Forms a Stacking Interaction during Binding and Destabilization of a Holliday Junction DNA Substrate

Yoshikawa

Iwasaki

Shinagawa

2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Escherichia coli RuvC resolvase is a specific endonuclease that recognizes and cleaves Holliday junctions formed during homologous recombination and recombinational repair. This study examines the phenotype of RuvC mutants with amino acid substitutions at phenylalanine 69 (F69L, F69Y, F69W, and F69A), a catalytically important residue that faces the catalytic center of the enzyme. F69Y, but not the other three mutants, almost fully complements the UV sensitivity of a ⌬ruvC strain and substantially resolves synthetic Holliday junctions in vitro. In the presence of 100 mM NaCl, RuvC F69A and F69L are defective in junction binding, but F69Y and F69W retain near wild-type binding activity during a gel shift binding assay. KMnO 4 was used to probe synthetic Holliday junction DNA in a complex with wild-type and mutant RuvC; F69A and F69L did not induce disruption of base pairing at the crossover to the same extent as wild-type RuvC. Thus, the aromatic ring of Phe-69 is involved in DNA binding, probably via a stacking interaction with a nucleotide base, and this interaction may induce a structural change in junction DNA that is required to form a catalytically competent complex.

show abstract

Section: Dna Repair Activities Of Phe-69 Mutants Of Ruvc-ruvc Mutantssupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 85%

mentioning

confidence: 99%

See 2 more Smart Citations

Evidence that Phenylalanine 69 in Escherichia coliRuvC Resolvase Forms a Stacking Interaction during Binding and Destabilization of a Holliday Junction DNA Substrate

Yoshikawa

Iwasaki

Shinagawa

2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Consistent with this mechanism of cleavage, the crystal structure of E. coli RuvC shows that the two monomers are related by a dyad axis, in which the two DNA binding clefts are separated by ∼30 Å ( Fig. 2A) (22,23). The 3D structure of Thermus thermophilus RuvC bound to an HJ has also been solved and confirms the presence of a twofold symmetric unfolded junction…”

supporting

confidence: 57%

Resolution of single and double Holliday junction recombination intermediates by GEN1

Punatar

Martín

Wyatt

et al. 2017

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

View full text Add to dashboard Cite

Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicaseTopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5′-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase, Escherichia coli RuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5′-A / T TT G / C -3′.

show abstract

Crystal Structure of an Archaeal Intein-encoded Homing Endonuclease PI-PfuI

Ichiyanagi

Ishino

Ariyoshi³

et al. 2000

Journal of Molecular Biology

109

View full text Add to dashboard Cite

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

Cited by 19 publications

References 44 publications

Evidence that Phenylalanine 69 in Escherichia coliRuvC Resolvase Forms a Stacking Interaction during Binding and Destabilization of a Holliday Junction DNA Substrate

Evidence that Phenylalanine 69 in Escherichia coliRuvC Resolvase Forms a Stacking Interaction during Binding and Destabilization of a Holliday Junction DNA Substrate

Resolution of single and double Holliday junction recombination intermediates by GEN1

Crystal Structure of an Archaeal Intein-encoded Homing Endonuclease PI-PfuI

Contact Info

Product

Resources

About