Lambda Int protein bridges between higher order complexes at two distant chromosomal loci attL and attR

Abstract

The excisive recombination reaction of bacteriophage lambda involves a specific and efficient juxtaposition of two distant higher order protein-DNA complexes on the chromosome of Escherichia coli. These complexes, which mediate synapsis and strand exchange, consist of two DNA sequences, attL and attR, the bivalent DNA binding protein Int, and the sequence-specific DNA bending proteins, IHF, Xis, and Fis. The protein-protein and protein-DNA interactions within, and between, these complexes were studied by various biochemical techniques and the patterns of synergism among pairs of mutants with marginally impaired recombination function were analyzed. The DNA bending proteins facilitated long-range tethering of high- and low-affinity DNA sites by the bivalent Int protein, and a specific map is proposed for the resulting Int bridges. These structural motifs provide a basis for postulating the mechanism of site-specific recombination and may also be relevant to other pathways in which two distant chromosomal sites become associated.

Fig. 1

Protein binding sites in attL and attR. The attL and attR sites are separated by 40,000 bp and comprise the junctions between phage DNA (straight line) and bacterial DNA (wavy lines). Nucleotide 0 is placed within the 7-bp overlap region, and the coordinates have positive numbers to the right and negative numbers to the left (46). The arm-type sites (ovals), bound by the NH₂-terminal domain of Int, are labeled P1 and P2 in attR and P′1, P′2, and P′3 in attL. The core-type sites (large arrows), bound by the COOH-terminal domain of Int, are labeled C and B′ in attR and B and C′ in attL. Those Int binding sites required for excisive recombination are denoted by solid ovals and arrows; unfilled ovals denote arm-type Int sites used only for integrative recombination (24, 32, 47). Binding sites for the DNA bending proteins are indicated by rectangles: the IHF binding sites (vertical lines) are H′ in attL and H1 and H2 in attR, the H1 site is used only for integrative recombination and its occupancy by IHF inhibits excisive recombination (39, 48); the Xis binding sites (rising slashes) are X1 and X2; the Fis binding site, F (falling slashes), overlaps the X2 site. Int binding at P2 is promoted by cooperative interactions with Xis at X1X2 (12, 24). The relative orientations of the DNA binding sites are indicated by arrows.

Fig. 2

IHF and Xis dependence of Int cleavage at the core sites of attR. (Top) Diagram of how the attR suicide substrates lead to formation of covalent Int-DNA complexes after Int cleavage at the C core site (top-strand nicked substrate) or at the B′ core site (bottom-strand nicked substrate). The 5′ terminus of the nicked strand was labeled with ³²P (49). (Bottom) Gel electrophoresis of the reactions of top- or bottom-strand nicked suicide substrate in the presence (+) or absence (−) of indicated proteins (49). The gel mobility of covalent complex (Int-attR) and attR suicide substrates is shown in the right margin. DNA fragments generated during the preparation of the suicide substrates are indicated as con.

Fig. 3

P2 dependence of Int cleavage and top-strand transfer. (A) Gel electrophoresis of covalent Int-DNA complexes at the C site of a half-attR with a wild-type (P2⁺) or P2-deleted (P2Δ) arm-type site, in the presence (+), or absence (−), of indicated proteins (49). The mobilities of the half-attR and Int-DNA complexes are indicated in the left margin. (B) Gel electrophoresis of a top-strand transfer assay for the P2⁺, or the P2-deleted, half-attR (³²P-labeled) with wild-type attL. The product of top-strand transfer is indicated in the right margin (rec) and its structure is shown (28). These reactions contain 0.1 pmol of at L DMA instead of the pBR322 carrier DMA (49).

Fig. 4

Gel retardation of attR higher order complexes. An attR (extending from the P2 site to the B′ core site) was incubated with the indicated proteins to generate complexes containing IHF (RH), Xis (RX), and IHF plus Xis (RHX). Complexes RHX1–RHX3 were generated by the addition of the indicated amounts of Int to complex RHX (50).

Fig. 5

A model for the Int-mediated synaptic interactions between the arm and core sites of attL and attR during excisive recombination. Bends in the attR (blue) and attL (orange) DMA are induced by IHF and Xis. The bivalent Int protein (green) forms intra-and intermolecular bridges: binding to the high-affinity arm sites (P′1, P′2, and P2) is via the smaller NH₂-terminal domain (small circle) and binding to the low-affinity core sites (B, B′, C, and C′) is via the larger COOH-terminal domain (large circle), which also contains the topoisomerase function. The relative positions of proteins and DMA in space and the relative order of interactions in time are not in the model.