Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

Whang, Ilson; Lee, Jehee; Jayaram, Makkuni

doi:10.1128/mcb.14.11.7492

Cited by 15 publications

(11 citation statements)

References 23 publications

(23 reference statements)

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“…2, lane 23). This result was not unexpected, based on previous observations that Flp(H305L) was much less active relative to Flp on altered substrates such as half-sites, full-sites with mismatches adjacent to the cleavage positions, or full-sites containing mismatched insertions within the spacer (Serre et al 1992;Whang et al 1994;C. Harkey and M. Jayaram, unpubl.…”

Section: Strand Cleavage In Synthetic Full Sites Containing Strand-spmentioning

confidence: 99%

Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity

Lee¹,

Tonozuka²,

Jayaram³

1997

Genes Dev.

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The Flp site-specific recombinase assembles its active site by recruiting the catalytic tyrosine (Tyr-343) from one Flp monomer into the pro-active site containing a triad of Arg-191, His-305, and Arg-308 from a second monomer. In principle, two active sites may be assembled from a Flp dimer by simultaneous, reciprocal contribution of the shared amino acids by its constituent monomers. In practice, only one of the two active sites is assembled at a time, as would be consistent with a recombination mechanism involving two steps of single-strand exchanges. By using substrates containing strand-specific base bulges, we demonstrate that the relative disposition of their DNA arms can account for this active site exclusion. We also show that the exclusion mechanism operates only at the level of positioning Tyr-343 with respect to the pro-active site, and not at the level of orienting the labile phosphodiester bond within the DNA chain. It is not negative cooperativity of substrate binding but, rather, the substrate-induced negative cooperativity in protein orientation that accomplishes half-of-the-sites activity in the Flp system.[Key Words: DNA recombination; active site assembly; nucleotide bulges; DNA conformation; catalytic complementation] Received July 17, 1997; revised version accepted September 5, 1997.Achieving spatial and temporal coordination of chemical bond breakage and formation is a challenge faced by multisubunit enzyme systems that accomplish one round of the final reaction through more than one chemical step. Phosphoryl transfer reactions in nucleic acids, RNA splicing, DNA transposition, and DNA recombination present this case. The crux of the problem is exemplified by conservative, site-specific recombination between two DNA partners, in which four chemically identical phosphodiester bonds are broken and joined by four subunits of the recombinase enzyme. The only biologically meaningful outcome is determined by a unique configuration of the breakage-joining events.Two pathways of conservative site-specific recombination have been shown (Stark et al. 1992;Jayaram 1994;Sadowski 1995). Recombinases that belong to the invertase/resolvase family make double-strand breaks in partner substrates and exchange strands in a concerted onestep reaction. Recombinases of the integrase (Int) family make single-strand cuts and complete recombination in two steps of single-strand exchanges. A Holliday junction is therefore an obligatory intermediate during Int family recombination. In both types of recombination, strand cleavage and strand joining are transesterification reactions, the former being executed by a recombinasederived nucleophile (an active site serine or tyrosine) and the latter by a DNA-derived nucleophile (a 5Ј or 3Ј hydroxyl group exposed by strand breakage). The conservation of the phosphodiester bond during strand cutting, by linkage of the broken DNA end to the recombinase, eliminates the requirement for an exogenous energy source for the progression of the reaction. The reaction proceeds without...

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Section: Strand Cleavage In Synthetic Full Sites Containing Strand-spmentioning

confidence: 99%

Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity

Lee¹,

Tonozuka²,

Jayaram³

1997

Genes Dev.

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“…The conditions for resolution were essentially the same as those for recombination reactions described previously (Chen et al 1992;Whang et al 1994). Normally, the reactions contained ∼0.025 to 0.05 pmole of the substrate and an excess of Flp (8-10 pmoles of Flp per pmole of Flp-binding element) in a reaction volume of 30 µl.…”

Section: Resolution Of Holliday and Y Junction Substratesmentioning

confidence: 99%

A tetramer of the Flp recombinase silences the trimers within it during resolution of a Holliday junction substrate

Lee

Jayaram²

1997

Genes Dev.

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Recombination catalyzed by the Flp site-specific recombinase involves breakage and joining of four DNA strands between two target substrates. The reaction is carried out in two steps of pairwise strand exchanges by a DNA-protein assembly in which four Flp monomers act cooperatively to execute strand cleavage and joining. Two models for recombination have been proposed. In the trimer model, the two active sites required for each step are assembled from three Flp monomers. In the tetramer (or dimer of asymmetric dimers) model, the two active sites are assembled from four Flp monomers, two monomers each contributing one active site. Experiments in which the two models challenge each other reveal that, within the Flp tetramer arranged on a Holliday junction, the two active sites required for its resolution are derived from all four, rather than three, Flp monomers. Thus, the relative protein subunit configuration of the tetramer silences the trimers within it by excluding them from assembling a functional active site pair.

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“…However, the assay buffer can eliminate DNase activity because the buffer contains high concentration of EDTA and is Mg +2 -free. Known integrases do not require Mg +2 as a cofactor (Whang et al, 1994), so presumably catalytic antibodies/Fabs with integrase activity will also not require Mg .…”

Section: Bmentioning

confidence: 99%

Development of Integrase-Like Catalytic Antibodies for Breast Cancer

Thompson¹

2001

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Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

Cited by 15 publications

References 23 publications

Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity

Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity

A tetramer of the Flp recombinase silences the trimers within it during resolution of a Holliday junction substrate

Development of Integrase-Like Catalytic Antibodies for Breast Cancer

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