Mutations in the amino-terminal domain of lambda-integrase have differential effects on integrative and excisive recombination

Abstract

Lambda integrase (Int) forms higher-order protein-DNA complexes necessary for site-specific recombination. The carboxy-terminal domain of Int (75-356) is responsible for catalysis at specific core-type binding sites whereas the amino-terminal domain (1-70) is responsible for cooperative arm-type DNA binding. Alanine scanning mutagenesis of residues 64-70, within full-length integrase, has revealed differential effects on cooperative arm binding interactions that are required for integrative and excisive recombination. Interestingly, while these residues are required for cooperative arm-type binding on both P'1,2 and P'2,3 substrates, cooperative binding at the arm-type sites P'2,3 was more severely compromised than binding at arm-type sites P'1,2 for L64A. Concomitantly, L64A had a much stronger effect on integrative than on excisive recombination. The arm-binding properties of Int appear to be intrinsic to the amino-terminal domain because the phenotype of L64A was the same in an amino-terminal fragment (Int 1-75) as it was in the full-length protein.

Fig. 1

Location and occupancy of protein-binding sites during integrative and excisive recombination. Integrative recombination between the phage attP and bacterial attB sites requires the phage-encoded Int protein and the host encoded IHF to generate the attL and attR prophage sites. Excisive recombination between attL and attR additionally requires the phage-encoded Xis protein and is assisted by the host-encoded Fis protein. Protein binding sites in the arms of attP, attL and attR that are occupied during integration and excision are shown as filled symbols. Open symbols represent unoccupied sites. Four core-type sites (C, C′, B and B′) flank two 7 bp overlap regions (O) as inverted repeats (inverted open arrows) and encompass each of the DNA-cleavage sites (curved arrows). There are five arm-type Int-binding sites (circles): two single sites in the P arm (P1 and P2) and three adjacent sites in the P′ arm (P′1, P′2 and P′3). IHF has three binding sites, H1, H2 and H′ (squares), and Xis has two binding sites, X1 and X2 (triangles), one of which (X2) overlaps with the single Fis-binding site (F; not illustrated).

Fig. 2

Residues within the 64–70 region are required for cooperative arm-binding. Full-length Int, various mutant proteins were tested in gel shift assays for their abilities to bind to the following arm-type DNA substrates; (A,B) 50 bp P′1,2, (C) 50 bp P′2,3 double arm-type sites and (D) 40 bp P′1, P′2 and P′3 single arm-type sites. The indicated concentrations of proteins were mixed with 50 nM and 10 nM radiolabelled DNA substrate for the single and double arm-type substrates, respectively, and incubated at 19°C for 30min. Reactions were analysed by electrophoresis on native 8% polyacrylamide gels which were subsequently dried and visualized by autoradiography. The intensity of the bands was measured as described in Experimental procedures. For the double and single arm-type sites the ratio of the doubly bound species to the total amount of substrate bound and the total amount of substrate bound has been calculated, respectively, and plotted as a function of protein concentration. Results are representative of at least three independent experiments.

Fig. 3. The defect in cooperative arm-binding of L64A is independent of the carboxy-terminus

A. Int 1–75 (N1–75) and Int 1–75L64A (N1–75L64A) proteins were tested in a gel-shift assay for their abilities to bind to arm-type DNA sites. A total of 200 nM radiolabelled 50 bp DNA substrate containing the two adjacent P′1,2 sites was incubated with the indicated concentrations of proteins at 19°C for 30min. B. Full-length wild-type and L64A proteins were tested in a gel shift assay for their abilities to bind to core-type substrate. In total, 100 nM radiolabelled 35 bp COC′ substrate was incubated with the indicated concentrations of protein at 19°C for 30min. C. Full-length wild-type and L64A were tested for their abilities to cleave a nicked COC′ suicide substrate. The indicated concentrations of protein were mixed with 5 nM radiolabelled DNA substrate and incubated at 25°C for 30min. The percent cleavage of the nicked COC′ suicide substrate is plotted as a function of protein concentration. D. Full-length wild-type and L64A proteins were tested in a gel-shift assay for their abilities to bind to a 40 bp duplex containing the P2X1 binding sites. The substrate DNA (13 nM) was preincubated at 19°C for 10 min with Xis (200 nM). Wild-type and L64A proteins (25 nM) were then added to the first tube followed by five serial two-fold dilutions and the reactions incubated at 19°C for 30min. Results are representative of at least two independent experiments.

Fig. 4

Two-distinct binding sites reside within the amino-terminus of Int. The chitin-binding domain (CBD) and the CBD–Int amino-terminal fusions (CBD–Int 1–62 and CBD–Int 1–75) were used to affinity precipitate full-length Int or C75, an amino-terminus-truncated Int as described in Experimental procedures. Briefly, reaction mixtures containing the appropriate Int protein, full-length wild-type or C75, were incubated with either the CBD alone or a CBD–Int fusion. The reactions were incubated at 4°C for 2.5 h and the CBD complexes precipitated via the addition of chitin. The precipitates were subsequently washed and loaded onto denaturing 12% SDS-PAGE gels. The proteins in the gel were subsequently transferred to PVDF membrane and analysed via Western blotting with (A) polyclonal rabbit anti-Int and (B) polyclonal rabbit antichitin binding domain antibodies. Results are representative of at least three independent experiments.

Fig. 5

L64 is required for both excisive and integrative recombination. Recombination reactions were carried out as described in the Experimental procedures. Briefly, reaction mixtures containing the appropriate substrates (A) supercoiled attR and linear attL and (B) supercoiled attP and linear attB were incubated with full-length wild-type, L64A, or R67A Int. The reactions were allowed to proceed for 1 h at 25°C before being analysed by agarose gel electrophoresis. The percent recombination is plotted as a function of protein concentration. Results are representative of two independent experiments.