Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli

Abstract

The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I- Sca I promoting intron mobility and as a maturase promoting intron splicing. Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I- Sca I endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli. We have also determined the cleavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3'-overhangs. Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16-19 bp sequence from positions -11 to +8 with respect to the intron insertion site.

Figure 1

Strategy for synthesis and sequence of a universal code equivalent for the ORF encoded by the second intron of the cytb gene of S.capensis. (A) (Top) Schematic of the orientation and overlapping of the oligonucleotides used for ORF synthesis; the 5′- and 3′-ends of the oligonucleotides are numbered according to their positions in the synthesized sequence presented in (B). (Bottom) Positions of the unique restriction sites present in the synthesized ORF. (B) Sequence of the coding strand with the sequence of the translated protein in the one letter code (amino acids in bold were specified by the non-universal codons in the mitochondrial sequence); restriction sites in the flanking regions used for cloning are in italic; changes introduced in the codons relative to the mitochondrial sequence are shown in lower case; brackets indicate the 5′-end of the sequences used to construct the p28bi2 and p25bi2 truncated forms of the endonuclease; the two conserved dodecapeptide (or LAGLIDADG) motifs are underlined.

Figure 2

SDS–PAGE of purified p28bi2 and p25bi2 truncated forms of the I-ScaI endonuclease.

Figure 3

p28bi2 endonuclease activity assays. (A) Structure of the plasmid pYGT28 used as substrate. pYGT28 was linearized with SspI before cleavage by p28bi2, which generates two DNA fragments of 2094 and 613 bp, respectively. (B) Lanes 1–4, 200 ng of substrate were incubated in the standard solution at 30°C for 30 min with 0.5, 1.0, 1.5 and 2 U p28bi2, respectively; lane 0, conditions as in lane 4 but Mg²⁺ was omitted. Optimal cleavage efficiency 90% (lane 4).

Figure 4

Determination of the optimal conditions for substrate cleavage by p28bi2. The graph shows the effects of (A) temperature, (B) pH, (C) MgCl₂ concentration and (D) NaCl concentration on the cleavage reaction. The conditions of the assays are described in Materials and Methods.

Figure 5

Velocity of cleavage reactions using the supercoiled or the linearized substrate. Supercoiled (open circles) or linearized (closed circles) pYGT28 (2.5 µg) was incubated with 10 U p28bi2 in 400 µl of standard solution at 30°C. Aliquots (40 µl) were taken at selected time points, the reactions were stopped and the products analyzed as described in Materials and Methods.

Figure 6

Stabilization of p28bi2 by interactions with DNA or RNA. p28bi2 (2 U) was incubated at 30°C for 10 min in the standard solution minus Mg²⁺ either without DNA or with 300 ng of the specific or non-specific substrate. After this preincubation, 8 mM Mg²⁺ and 300 ng of substrate pGYT28 were added to all the assays except the reaction shown in lane 3, where only the addition of Mg²⁺ was necessary, then assays were incubated for a further 30 min at 30°C, stopped and analyzed. Lane 1, standard reaction shown as reference (300 ng of pYGT28 substrate and 2 U enzyme); lane 2, preincubation without DNA; lane 3, with pYGT28; lane 4, with linearized plasmid pET22 (5.4 kb); lane 5, with the 30 nt long oligoduplex; lane 6, with the 26 nt RNA stem–loop.

Figure 7

Determination of the cleavage site. Autoradiogram showing the extension polymerization products digested or not with p28bi2 and the corresponding sequence ladder. The cleavage sites are indicated by arrows.

Figure 8

Mutational analysis of the p28bi2 recognition sequence. The wild-type sequence of the target site is shown on top with the cleavage site indicated by a staggered line and the intron insertion site by an arrow. The three possible base substitutions for each position are indicated on the left side of the grid. closed circles, the wild-type sequence; +, mutant cleaved as well as the wild-type; ɛ, reduced cleavage; –, no cleavage.