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Figure 3 | Mobile DNA

Figure 3

From: The site-specific integration reaction of Listeria phage A118 integrase, a serine recombinase

Figure 3

In vitro recombination by A118 integrase. (A) Integrative recombination between a 4.8 kb supercoiled plasmid containing attP (pRJ2214) and a 100 bp attB fragment generates a 4.9 kb linear product. (B) Agarose gel showing a time course of intermolecular recombination between supercoiled attP and linear attB substrates. (C) Titration of increasing amounts of the attB fragment relative to the supercoiled attP plasmid on rates of intermolecular recombination. Molar ratios of attB to attP (set at 1) are given. (D) Intramolecular deletion reaction between attP and attB on pBCPB-A1+ generates 3.5 and 3.9 kb deletion products that are linearized after digestion with Bam HI and Xho I, respectively. (E) Deletion reactions between attP and attB (pBCPB-A1+) or between attL and attR (pRJ2913; no recombinant products formed). Integrase reactions were for 40 minutes. (F) Deletion reactions (attP×attB) performed in the absence or presence of the strong metal chelator neocuproine. Integrase reactions were for 40 minutes. (G) Recombination rates in the presence of chelators. Deletion reactions were performed using integrase from storage buffer with 0.1 mM ethylenediamine tetraacetic acid (control, dark blue) or pre-incubated at 4°C overnight with 50 mM EDTA (red) or 20 mM neocuproine (green) in buffer without added metal. Final concentrations of EDTA and neocuproine in the reactions were 5 and 2 mM, respectively. (H) Recombination rates in reactions supplemented with metals and polyamines. Supplements were added to 10-minute deletion reactions with or without 5 mM spermidine. Supplements (MgCl2, CaCl2, BaCl2, MnCl2, CsCl, and NiCl2) were at 10 mM except for ZnSO4, which was at 1 mM because higher amounts were inhibitory; FeCl2, CoCl2, CuCl2 at 1 mM were inhibitory. Recombination rates (% deletions in 10 minutes, average of at least three experiments) are given relative to the unsupplemented reaction (control) in the presence of spermidine, which was set to 1.

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