The Orientation of Mycobacteriophage Bxb1 Integration Is Solely Dependent on the Central Dinucleotide of attP and attB  Pallavi Ghosh, Amy I Kim, Graham F Hatfull  Molecular Cell  Volume 12, Issue 5, Pages 1101-1111 (November 2003) DOI: 10.1016/S1097-2765(03)00444-1

Figure 1 Bxb1 attP and attB Site Requirements for Integrative Recombination (A) Bxb1 attP and attB sites share an 8 bp common core (shown in lower case) flanked by inverted repeats. Base pairs that are present in symmetrical positions are shown in bold and by horizontal arrows; vertical arrows show putative scissile bonds; the minimum lengths required for recombination are boxed. Minimal length requirements of attP (B) and attB (C) were determined using scDNA and dsDNA oligonucleotide linear substrates in in vitro recombination reactions. Positions of supercoiled substrates (sc) and linear products (lin) are indicated; small linear oligonucleotide substrates are not seen. The ratios of oligonucleotide:plasmid DNAs are 5:1 (B) and 4:1 (C). The lengths of the DNA substrates tested are shown above each lane; the center of the sites shown in (A) is at the center of each substrate, each of which represents progressive deletion of the substrates by 1–2 bp at each end. The reaction scheme, which involves integrative recombination between a supercoiled DNA (scDNA) substrate containing one site (either attP or attB) and a short oligonucleotide-derived dsDNA substrate containing the partner site, is shown in (D). Molecular Cell 2003 12, 1101-1111DOI: (10.1016/S1097-2765(03)00444-1)

Figure 2 DNA Cleavage by Bxb1 gpInt (A and B) A 376 bp Hind III-Bam HI attB DNA fragment, labeled at either the Hind III (A) or Bam HI (B) ends, was incubated with either gpInt or a catalytically defective mutant of gpInt (S10A), unlabeled wild-type or mutant (G4C) partner DNA substrates (attP in A and attB in [B], both 50 bp), and 40% ethylene glycol, as indicated. Reactions were incubated for 1 hr at 37°C, and the products separated on an 8% polyacrylamide gel. Where indicated, samples were treated with proteinase K prior to electrophoresis. The mobility of the free DNAs, DNA-gpInt complexes, and dsDNA cleavage products are indicated. dsDNA cleavage products (*–) are observed both in the presence of 40% ethylene glycol and when mismatched partner DNA is included. Bands indicated as *−gpInt are putative protein-DNA complexes in which gpInt is covalent attached to DNA cleaved at the crossover site. The horizontal arrows indicate a slow-migrating band that may represent a synaptic complex containing attP and attB. Alternatively numbered lanes are shown at the bottom of these panels. The sizes of product DNAs and cleavage products are as follows: attB, 376 bp; attL, 224 bp; attR, 202 bp; cleavage products, 199 bp and 177 bp. A similar pattern of complexes, recombinant products, and cleavage products is observed using radiolabeled attP DNA and unlabeled attB DNA (data not shown). (C) shows an identical similar set of reactions to those in (A) (but without lane 1, and including a dideoxy sequencing ladder using a primer with the same 5′ end as the labeled DNA), separated on a 5% denaturing polyacrylamide gel. The lanes are numbered in accord with the reactions in (A). The exact positions of the cleavage sites are shown in (D), where cleavage products (Rx; generated as in [A], lane 16) are sized using didexoy sequencing ladders made with primers having identical 5′ ends to the end-labeled DNA fragments. The sequence of the 8 bp common core (numbered accordingly) and the cleavage sites are shown below. Molecular Cell 2003 12, 1101-1111DOI: (10.1016/S1097-2765(03)00444-1)

Figure 3 Bxb1 Integrase Catalyzes Both Recombination and Topological Relaxation (A and B) Recombination reactions between scDNA and an oligonucleotide partner were performed for varying times (as shown) and the products separated by agarose gel electrophoresis in the presence of ethidium bromide (EtBr). The starting molar ratio of attB:attP DNA in both sets of reactions is approximately 1:1. Positions of supercoiled substrate DNA (sc sub.) and linear products (lin. prod.) are indicated. The band migrating slightly faster than the supercoiled substrate DNA (circ. prod.) is likely a circular product that is a topologically closed relaxed form of the substrate DNA that contains few supercoils (prior to exposure to ethidium bromide during electrophoresis). (C) Reactions similar to those in (A) were analyzed by gel electrophoresis in the absence of EtBr. Note that the putatively topologically closed product circles (circ. prod.) migrate slower than linear product DNA (lin. prod.) in the absence of ethidium bromide, consistent with these containing few supercoils. The attB:attP ratio is 4:1. The bands seen between the supercoiled DNA substrate and the linear product, and between the linear product and the circular product, are likely topoisomers of the substrate DNA with fewer supercoils than the starting material. (D) Recombination reactions containing supercoiled attP DNA and varying amounts of attB were incubated for 2 hr and examined by agarose gel electrophoresis without EtBr; attB:attP ratios are 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 10:1, 100:1, 1000:1. Bands are labeled as in (C). (E) Time course of integration reactions in the absence of attB DNA, showing the dependence upon partner DNA for topological relaxation. Open circles (oc), supercoiled substrate (sc. sub) DNA, and the position of the linear product (lin. prod.) are indicated. Molecular Cell 2003 12, 1101-1111DOI: (10.1016/S1097-2765(03)00444-1)

Figure 4 Role of the Central Dinucleotide (A) Recombination reactions were performed with supercoiled attP DNA and small linear attB substrates containing base substitutions at each of the 8 bp of the common core and the products analyzed on a 0.8% agarose gel lacking ethidium bromide. Substitutions at positions 1–3 and 6–8 (see Figure 2D for numbering scheme) have little effect on recombination, whereas changes at the central dinucleotide (positions 4 and 5) inhibit recombination while promoting topological relaxation of attP DNA. The attB:attP molar ratios are 1:1. (B) Time course of relaxation using a T5C attB mutant and wild-type attP showing progressive formation of topoisomers. Subsequent addition of wild-type attB DNA after 120 min shows that the products of relaxation act as substrates for further rounds of recombination. (C) Central dinucleotide homology restores recombination and substitution with G4C, T5A or T5C in both recombination partners enables recombination. The attB:attP molar ratio in (B) and (C) is 4:1. Molecular Cell 2003 12, 1101-1111DOI: (10.1016/S1097-2765(03)00444-1)

Figure 5 Substrates with Palindromic Central Dinucleotides Lose Orientation Specificity (A) 5′-GC central dinucleotides in both attP and attB (T5C x T5C) results in formation of equal amounts of normal products (i.e., attL & attR) and products in which the left half of attP is joined to the left half of attB (BP) and the right halves of attP and attB are joined (B′P′); only the expected products are formed with non-palindromic central dinucleotides (A). Correct (attL & attR) and aberrant products (BP and B′P′) are formed with similar kinetics when the T5C substitution is at both sites (B). Both attP and attB substrates are scDNA and reactions were digested with PstI prior to electrophoresis. DNA size markers (M) correspond to 10, 8, 6, 5, 4, and 3 kbp fragments. The sizes of substrates and products are as follows: attP, 4.8 kbp; attB, 3.5 kbp; attL, 7.7 kbp; attR, (643 bp); BP, 4.5 kbp; B′P′, 3.8 kbp. The small attR-containing product is not shown. Molecular Cell 2003 12, 1101-1111DOI: (10.1016/S1097-2765(03)00444-1)

Figure 6 In Vivo Recombination of Central Dinucleotide Mutant Substrates (A)Plasmids pMol2 and pMol1 contain attP and attB sites in direct and inverted orientations, respectively. When transformed into a strain of E. coli expressing Bxb1 gpInt, pMol2 is efficiently resolved to give a deletion derivative that is sensitive to hygromycin; pMol1 is efficiently inverted and transformants are hygromycin resistant. Plasmids containing central dinucleotide substitutions in both attP and attB are otherwise identical to pMol2 (B) Behavior of altered central dinucleotide substrates in E. coli. Following electroporation with various plasmids, transformants were recovered on media containing either hygromycin and chloramphenicol, or just chloramphenicol. With the exception of the 5′-GC mutant, direct plating of the transformation mix yielded few hygromycin resistant transformants (<1%) when the sites are in direct orientation and approximately equivalent numbers when in inverted orientation (column 3). For the 5′-GC mutant (direct orientation) approximately equal numbers of transformants were obtained; 181 colonies selected on chloramphenicol alone were screened for their hygromycin phenotype. Five colonies (3%) were only weakly resistant to hygromycin and when the plasmids were recovered and analyzed, contained a mixture of recombined and unrecombined DNAs. In all other cases, DNA from at least 15 individual transformants was recovered and analyzed by restriction. In every case hygromycin sensitivity correlated with deletion, and hygromycin resistance with inversion (column 4). No transformants carrying only unrecombined DNA were identified with any plasmid. Molecular Cell 2003 12, 1101-1111DOI: (10.1016/S1097-2765(03)00444-1)

Figure 7 Recombination Reactions between Singly Methylphosphonate-Substituted attB DNAs and Supercoiled attP DNA Recombination reactions were performed between either supercoiled wild-type attP DNA (B) or supercoiled mutant attP (T5C) (C) and methylphosphonate-substituted attB DNA substrates, and analyzed by agarose gel electrophoresis. Each reaction was performed with attB:attP ratios of either 4:1 or 0.5:1 for 1 hr. Substitution at the scissile bonds in attB inhibits both recombination and topological relaxation of attP DNA. The positions of methylphosphonate substitutions are shown in (A). Molecular Cell 2003 12, 1101-1111DOI: (10.1016/S1097-2765(03)00444-1)