Polynucleotidyl transfer reactions in site‐specific DNA recombination

Mizuuchi, Kiyoshi

doi:10.1046/j.1365-2443.1997.970297.x

Cited by 136 publications

(114 citation statements)

References 81 publications

(103 reference statements)

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“…9,10,13 This 3 0 -processing forms hydroxyl groups necessary for integration. 9,10,14 IN then facilitates strand transfer through a one-step mechanism that both induces a staggered break in the phosphodiester bonds of the target DNA and joins a single vector DNA end to the target DNA through a transesterification reaction. 11,14,15 Once both vector DNA ends are attached, host enzymes repair the resulting gapped intermediate, generating an integrated provirus ( Figure 2).…”

Section: Transduction Biologymentioning

confidence: 99%

Integrase-defective lentiviral vectors: progress and applications

2009

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Lentiviral vectors (LVs) offer the advantages of a large packaging capacity, broad cell tropism or specific cell-type targeting through pseudotyping, and long-term expression from integrated gene cassettes. However, transgene integration carries a risk of disrupting gene expression through insertional mutagenesis and may not be required for all applications. A non-integrating LV may be beneficial in cases in which transient gene expression is desired. Several recent publications outline the development and initial biological characterization of such vectors. Here, we discuss the potential applications and new directions for the development of integration-defective LVs.

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Section: Transduction Biologymentioning

confidence: 99%

Integrase-defective lentiviral vectors: progress and applications

2009

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“…The four acidic residues Asp-7, Glu-66, Asp-138, and Asp-141 constitute the catalytic center of RuvC (21). These residues coordinate divalent metal cation(s) such as Mg 2ϩ or Mn 2ϩ and make a pentacoordinated intermediate using the metal ion(s) and phosphodiester bonds at the cleavage site on the continuous strand (17,21,33). Two basic residues, Lys-107 and Lys-118, interact with the negatively charged phosphate of the DNA backbone via electrostatic interactions and stabilize the pentacoordinated intermediate in which negative charge accumulates during the transition state of hydrolysis (22).…”

Section: Aromatic Side Chain Of Phe-69 Is Involved In Helix Destabilimentioning

confidence: 99%

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

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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.

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“…S equence-specific cleavage of double-stranded DNA underpins many genetic events, including recombination, transposition, viral DNA integration, and the restriction of DNA (1)(2)(3)(4). The enzymes that make double-strand breaks use many different strategies to achieve this end.…”

mentioning

confidence: 99%

How the Bfi I restriction enzyme uses one active site to cut two DNA strands

Sasnauskas

Halford

Šikšnys

2003

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

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Unlike other restriction enzymes, BfiI functions without metal ions. It recognizes an asymmetric DNA sequence, 5 -ACTGGG-3 , and cuts top and bottom strands at fixed positions downstream of this sequence. Many restriction enzymes are dimers of identical subunits, with one active site for each DNA strand. Others, like FokI, dimerize transiently during catalysis. BfiI is also a dimer but it has only one active site, at the dimer interface. We show here that BfiI remains a dimer as it makes double-strand breaks in DNA and that its single active site acts sequentially, first on the bottom and then the top strand. Hence, after cutting the bottom strand, a rearrangement of either the protein and͞or the DNA in the BfiI-DNA complex must switch the active site to the top strand. Low pH values selectively block top-strand cleavage, converting BfiI into a nicking enzyme that cleaves only the bottom strand. The switch to the top strand may depend on the ionization of the cleaved 5 phosphate in the bottom strand. BfiI thus uses a mechanism for making double-strand breaks that is novel among restriction enzymes.S equence-specific cleavage of double-stranded DNA underpins many genetic events, including recombination, transposition, viral DNA integration, and the restriction of DNA (1-4). The enzymes that make double-strand breaks use many different strategies to achieve this end. The simplest may be the orthodox type II restriction enzymes such as EcoRI or EcoRV, dimers of identical subunits that recognize palindromic DNA sequences. In the presence of Mg 2ϩ , they cleave both strands at fixed positions in their recognition sites (3). They bind their target sites symmetrically, so that one active site from the dimer is positioned against one DNA strand and likewise the second active site on the other strand. Independent reactions in each active site then generate the double-strand break. Some homing endonucleases, such as I-CreI, also act in this way (4), as do the enzymes that resolve Holliday junctions (2).This strategy cannot apply to enzymes that recognize nonpalindromic sites and cut both strands away from the recognition site (3). For example, FokI, a type IIS restriction enzyme, recognizes the sequence 5Ј-GGATG-3Ј and cuts top and bottom strands 9 and 13 nt downstream of this site, respectively (5). FokI is a monomer with separate domains for DNA recognition and catalysis. The catalytic domain has a single active site, which is like that in each subunit of an orthodox enzyme, so the FokI monomer cannot cleave both DNA strands (6). To make doublestrand breaks, the monomer of FokI bound to its recognition site associates transiently with a second monomer to form a dimer with two active sites (7-9). Many type IIS enzymes operate in this way (10,11).An alternative to using a homodimeric enzyme to make double-strand breaks is an enzyme with different subunits. For example, the transposition of Tn7 requires two proteins, TnsA and TnsB, that each cleave one strand of the DNA (12). Interestingly, the structure of TnsA is similar...

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Polynucleotidyl transfer reactions in site‐specific DNA recombination

Cited by 136 publications

References 81 publications

Integrase-defective lentiviral vectors: progress and applications

Integrase-defective lentiviral vectors: progress and applications

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

How the Bfi I restriction enzyme uses one active site to cut two DNA strands

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