Patterns of lambda Int recognition in the regions of strand exchange

Abstract

Int protein has two classes of binding sites within the phage att site: the arm-type recognition sequences are found in three specific sites that are distant from the region of strand exchange; the junction-type recognition sequences occur as inverted pairs around the crossover region in both attP and attB. During recombination between attP and attB each of the four DNA strands is cut at a homologous position within each of the junction-type Int binding sites. In all four junction-type sites Int protein interacts primarily with the same face of the DNA helix, as determined by those purine nitrogens that are protected against methylation by dimethylsulfate. Efficient secondary attachment sites for lambda contain sequences with partial homology to the junction-type binding sites. In addition, the sequence between, but not part of, the two junction-type sites (the overlap region) is strongly conserved in secondary att sites. Thus, in the vicinity of strand exchange, attP and a recombining partner, such as attB, are very similar; each comprises two junction-type Int recognition sites and an overlap (crossover) region.

Figure 1. Int Protection of Non-att Sequences Homologous to the attP Core Region

(A) The attP-containing fragment *Bam(+242)–Hind III(−250), from pWR1 (Hsu et al., 1980), partially digested with NCS in the presence (+) or absence (−) of Int. (* denotes the 5′-labeled end.) The P′ arm site and common core protected regions are visible further up the gel beyond the int155 site, which is marked by a bracket. Sequence of the 30 bp protected region at the int155 site is compared with that of the attP core region in Figure 2. (B) Fragment *Pst(3608)–Hinc II(3906) from pBR322 partially digested with DNAase I in the presence (+) or absence (−) of Int. (C) Fragment *Hinf(3361)–Pst(3608) from pBR322, partially digested with DNAase I in the presence (+) or absence (−) of Int. Sequences of the protected regions in (B) and (C) are illustrated in Figure 3A.

Figure 2. Comparison of Int-Protected Sequences in the attP Core Region and the int155 Site

Sequences have been previously reported (Landy and Ross, 1977). The regions of homology between the two sites, which constitute in each case an imperfect inverted repeat (→←), are represented in large letters. Approximate boundaries of NCS footprint protection are indicated (●). Sites in the central attP core that are sensitive to NCS are circled (Ross et al., 1982). No such sites are seen in the int155 region, but would not be expected since NCS is an A+T-specific reagent. ( ) Sites cut by Int during recombination (Mizuuchi et al., 1981; N. Craig and H. Nash, personal communication). The endpoints of xin-promoted deletions (▼) (Gritzmacher and Weisberg, personal communication) and the 8 bp region containing the att²501 deletion endpoint (Ross et al., 1982) are indicated within the int155 site.

Figure 3. Sequences of Int-Protected Junction-type Sites

All sequences are written 5′ to 3′. (A) Non-att junction-type sites. Protection profiles of pBR322 3511 and 3755 are shown in Figures 1B and 1C, and of “bio” in Ross et al. (1979). Sequences are aligned according to homology, and the numerical coordinates given are from the published sequences (“bio”—Landy and Ross, 1977; Ross et al., 1979; pBR322—Sutcliffe, 1979). Homologous positions are presented in large type. Approximate boundaries of footprint protection are indicated for DNAase I (○) and NCS (●). Definition of very precise boundaries is not possible because not all positions are good substrates for the reagents used (NCS is A+T-specific, see D’Andrea and Hazeltine, 1978). (B) Sequences of both left and right core–arm junctions from attP and attB aligned according to homology among them and homology with the non-att sites in (A). Numbering is from Landy and Ross (1977) and protection boundaries are from Ross et al. (1979). Boundaries of the right side of the two attP sites incorporate data from NCS-sensitive central core positions (Ross et al., 1982). (C) Sequences from the left and right sides of the int155 site. Regions of homology to other sites in (A) and (B) are in large letters. The pairs of sites in (B) and (C) are present in inverted orientation and with identical spacing in the attP, attB and int155 sites (see Figures 2 and 5).

Figure 4. Int Protection of the attP and attB Core Regions against Methylation by DMS

The attP core region fragment from pMJB11 (containing only the core Int binding site, but no arm binding sites, see Experimental Procedures) was 5′-labeled in the lower strand (Bam site at +46) (A), or the upper strand (Hind III site at −70) (B), and methylated in the presence (+) or absence (−) of Int (see Experimental Procedures). An attB core region fragment from pMM291 (containing attB sequence from −17 to +21; Mizuuchi et al., 1981) was 5′-labeled in the lower strand (Hha site at 4257 in pBR322) (C), or the upper strand (Hga I site at 390 in pBR322) (D), and methylated in the presence (+) or absence (−) of Int. C+T chemical sequence markers were prepared by procedures of Maxam and Gilbert (1980). Samples were electrophoresed in 10% acrylamide–8 M urea gels (see Ross et al., 1979, 1982). Positions protected by Int against methylation (*). Smaller (*) indicates slight degree of protection. Positions of enhanced methylation in the presence of Int (→). The outermost affected positions in each site are numbered; see Figure 5 for identification of other protected positions.

Figure 5. attP and attB Core Region Sequences Showing Methylation Protection or Enhancement in the Presence of Int

(*) Positions protected against methylation. Smaller asterisk indicates slight degree of protection. (↑) Positions showing enhanced levels of methylation. Sequences were reported previously (Landy and Ross, 1977). 0 is the center of the 15 bp common core sequence. Positions in larger letters are regions of homology between the left and right core–arm junctions (situated in inverted orientation (→←) and conserved in non-att junction-type binding sites (Figure 3). ( ) Positions cut by Int during recombination (Mizuuchi et al., 1981; Craig and Nash, personal communication). The four junction sites are referred to as C and C′ (for core) in attP and B and B′ in attB. Protected positions that are conserved in three or four of the sites (●).

Figure 6. Subcloned Left (pWR243) and Right (pWR221, pPH411) attP Core–Junction Binding Sites (C and C′)

Sequence conserved among junction-type binding sites (see Figure 3) is represented in larger letters. Purine positions protected by Int against methylation by DMS (*), or showing enhanced levels of methylation in the presence of Int (↑), are indicated (compare with Figure 5). Only the upper strand of pWR221 was tested. Boundaries enclosing sequences that are protected by Int in DNAase I (○) or NCS (●) footprint protection experiments are indicated. pWR243 shows no footprint protection under the conditions employed (see text). Protection of pWR221 is partial. See Experimental Procedures for clone constructions. The newly created junctions of att and pBR322 sequence are indicated ( ). In pWR243 and pWR221, these new junctions occur at the right or left ends of the Dde site (CTAAG). In pPH411, the new junction was created at the Hinc II site in pBR322 (3906) and the Alu site in att (−6); no new restriction site was generated, and one C was deleted during fusion formation. Numbering refers to att sequence (Figure 5) or to pBR322 sequence (Sutcliffe, 1979).

Figure 7. Int Recognition Sequences in Secondary att Sites

Sequence positions conserved with attP or attB, or non-aft junction-type binding sequences (Figure 3) are in large letters under the two regions marked INT. Comparison with the attP core junction sites (C and C′) is included since a subcloned attP core region functions well as a “recipient” site (attB analog) (Hsu et al., 1980; W. Bushman and A. Landy, unpublished observations). Positions of the 7 bp staggered cuts in upper ( ) and lower ( ) strands of attP and attB are indicated. The overlap region comprises the 7 bp between the cut positions. Secondary site sequences are aligned according to cut positions, as judged by sequence analysis of the secondary prophage sites (see Table 1 for references; sites are in the same order of measured or predicted efficiency as in Table 1). Many secondary sites contain several bases of continuous homology with the attP core, which may include one or both of the cut positions; in these cases, the cuts in the secondary sites are assumed to occur in the same relative positions as in the attP and attB cores. In some cases better sequence matches with the recognition site are found ±1 bp from the standard spacing (see Table 1), but are not indicated here. Sequence homologies in the overlap region are not given special representation, with the exception of the two positions (−1 and +3) that are conserved positions in the Int consensus sequence.

Figure 8. B-Form DNA Model of the attP Core Region from −15 to +15

The helix is oriented such that the left or “C” site occupies the lower part of the figure, and the right or “C′” site the upper part. Red atoms = oxygen; yellow = phosphorus; white = hydrogen; black = carbon; blue = nitrogen. Positions of phosphodiester linkages cut by Int during recombination (Mizuuchi et al., 1981; Craig and Nash, personal communication) are indicated by green arrows, with the lower arrow point situated directly below the top strand (−2/−3) linkage, and the upper arrow point directly above the bottom strand (+4/+5) linkage (refer to Figure 5). Purine positions N7 of guanine (major groove) or N3 of adenine (minor groove), at which levels of methylation are affected by bound Int, are marked with labeled yellow disks. All but one of the affected purine positions are on the front 180° of the helix, as shown, G (−13), on the rear of the helix, which shows enhanced methylation, is not visible in this view. Enhanced positions are indicated by vertical arrows on the yellow disks. Three positions in each site, marked additionally with green spots on the yellow disks, indicate conserved sequence positions in the two sites: (−9, −6, −5, in C; and +7, +8, +11, in C′; see also Figure 5).