Portal de Periódicos da CAPES

The isomeric preference of Holliday junctions influences resolution bias by lambda integrase

1997; Springer Nature; Volume: 16; Issue: 12 Linguagem: Inglês

10.1093/emboj/16.12.3744

1460-2075

Marco A. Azaro,

Photosynthetic Processes and Mechanisms

Article15 June 1997free access The isomeric preference of Holliday junctions influences resolution bias by λ integrase Marco A. Azaro Marco A. Azaro Division of Biology and Medicine, Brown University, Box G-J360, Providence, RI, 02912 USA Search for more papers by this author Arthur Landy Corresponding Author Arthur Landy Division of Biology and Medicine, Brown University, Box G-J360, Providence, RI, 02912 USA Search for more papers by this author Marco A. Azaro Marco A. Azaro Division of Biology and Medicine, Brown University, Box G-J360, Providence, RI, 02912 USA Search for more papers by this author Arthur Landy Corresponding Author Arthur Landy Division of Biology and Medicine, Brown University, Box G-J360, Providence, RI, 02912 USA Search for more papers by this author Author Information Marco A. Azaro1 and Arthur Landy 1 1Division of Biology and Medicine, Brown University, Box G-J360, Providence, RI, 02912 USA The EMBO Journal (1997)16:3744-3755https://doi.org/10.1093/emboj/16.12.3744 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info λ site-specific recombination proceeds by a pair of sequential strand exchanges that first generate and then resolve a Holliday junction intermediate. A family of synthetic Holliday junctions with the branch point constrained to the center of the 7 bp overlap region was used to show that resolution of the top strands and resolution of the bottom strands are symmetrical but stereochemically distinct processes. λ integrase is sensitive to isomeric structure, preferentially resolving the pair of strands that are crossed in the protein-free Holliday junction. At the branch point of stacked immobile Holliday junctions, the number of purines is preferentially maximized in the crossed (versus continuous) strands if there is an inequality of purines between strands of opposite polarity. This stacking preference was used to anticipate the resolution bias of freely mobile junctions and thereby to reinforce the conclusions with monomobile junctions. The results provide a strong indication that in the complete recombination reaction a restacking of helices occurs between the top and bottom strand exchanges. Introduction The site-specific recombination system of bacteriophage λ mediates insertion of the circular phage DNA into the Escherichia coli host chromosome, as first proposed by Allan Campbell (Campbell, 1962; reviewed in Landy, 1989; Stark et al., 1992). This is a highly programmed transaction that occurs between two loci, the 240 base pair (bp) phage attP and the 25 bp bacterial target attB. The products of this recombination are two hybrid sites, attR and attL, that form the boundaries of the integrated prophage. An excisive recombination can occur between these sites to regenerate the ‘substrates’, attP and attB. Both of these reactions are characterized by two pairs of temporally distinct strand exchanges, separated by 7 bp (overlap region), that proceed via an obligatory Holliday junction intermediate (Kitts and Nash, 1987; Nunes-Düby et al., 1987). In essence, this reaction can be said to convert continuous (i.e. stacked) parental helices into continuous recombinant helices; thus, the substrates are not only reshuffled but restacked. We wished to determine at what stage of the recombination reaction this restacking occurred and to test a previously proposed model for strand exchange (Nunes-Düby et al., 1995). The catalytic steps are executed by the phage-encoded λ integrase (Int, a type I topoisomerase) in concert with other protein factors (IHF for integrative recombination; IHF, Xis and Fis for excisive recombination). Two λ Int monomers introduce a nick into the top strand of each recombining partner at the 5′-boundary of the 7 bp overlap and in the process become covalently attached via a 3′-phosphotyrosyl bond (Mizuuchi et al., 1981; Craig and Nash, 1983; Pargellis et al., 1988). It has been proposed that, once the 5′-ends are freed by the nicks, the top strands of each overlap region swap 2–3 nt with the partner duplex (Nunes-Düby et al., 1995). If proper base pairing is achieved, the newly exchanged strands become ligated to the receiving strands, disconnecting the two λ Int monomers in the process. According to this model, a Holliday junction intermediate is created with its crossover sitting near the center of the overlap region. The conformation of the newly generated Holliday junction most closely reflects the helical axes of the parental duplexes. The model calls for a limited net movement of the branch point of ∼1–3 bp and for the Holliday junction to assume a different isomer, that now anticipates the helical axes of the recombinant helices. This isomerization (which constitutes the restacking step) prepares the Holliday junction for resolution by cleavage of the bottom strands. This model of λ site-specific recombination is based on studies of synthetic Holliday junctions that have their branch migration constrained by the incorporation of sequence heterologies (Figure 1; Nunes-Düby et al., 1995). It challenges the long-standing notion that the requirement for overlap sequence homology (Weisberg et al., 1983) is to permit branch migration across the full 7 bp of the overlap region. A critical conclusion drawn from that study is that the Holliday junctions are optimally resolved by λ Int when the branch point is located in a 1–3 bp region at the center of the 7 bp overlap region. Most strikingly, the DNA junctions are efficiently and exclusively resolved to ‘substrate’ helices (top strand resolution) when the branch point is fixed at position 3/4, immediately left of the center of the overlap region, and to ‘product’ helices (bottom strand resolution) when the branch point is fixed at position 4/5, immediately right of the center. In order to postulate that these reactions are symmetrical (i.e. that the geometries of top strand resolution and bottom strand resolution are identical), the authors invoked an isomerization step between the first and second pairs of strand exchanges. Figure 1.Strand swapping model of recombination as proposed by Nunes-Düby et al. (1995). In this representation the substrate helices (attP and attB or attL and attR) are aligned in an approximately antiparallel orientation (synapsis). λ Int nicks the top strands (red and yellow ribbons) at the top strand cleavage sites (red arrows). A short top strand segment (∼3 nt) disanneals from each original helix, exchanges and becomes ligated to the partner helix. A Holliday junction intermediate, in which the top strands are sharply bent at the branch point (TC isomer), is generated. This species isomerizes to a conformation that resembles a BC isomer and, in the process, the substrate helices are unstacked and then restacked into an orientation that anticipates the product helices. Notice that in this structure the bottom strands (gray and blue ribbons) are now sharply bent at the branch point. λ Int now cleaves, exchanges and ligates the bottom strands at the sites indicated by blue arrows to generate a pair of product helices (attL and attR or attP and attB). Download figure Download PowerPoint The structure of immobile four-way junctions in solution has been extensively characterized (reviewed in Lilley and Clegg, 1994; Seeman and Kallenbach, 1994) and has been shown to depend both on the ionic conditions and on the sequence at the branch point (Duckett et al., 1988, 1990). In the absence of metal ions the Holliday junction assumes an extended structure, in which the four helices are unstacked and point towards the corners of a square. Addition of micromolar concentrations of divalent cations, such as Mg2+, promotes pairwise stacking of the helical arms and rotation into an antiparallel X-structure. Millimolar concentrations of monovalent cations, such as Na+, can also stimulate this transition, albeit less efficiently. A consequence of this structural transition is to generate two strands that are ‘continuous’ with each helical axis and two strands that are sharply bent and ‘exchange’ between the two stacked helices. In this study we shall refer to the latter pair of strands as ‘crossed’, to avoid any confusion with strand exchange during resolution. It has also been shown that immobile four-way junctions choose one of two possible isomers of the stacked structure, based on their relative stability (Duckett et al., 1988). This stability is dictated, in an unknown manner, by the sequence of the base pairs at the branch point. The two possible stacking isomers are relevant to our recombination model in the following way: we had predicted that a Holliday junction with its top strands crossed will be resolved preferentially to parental helices and one with its bottom strands crossed will be resolved preferentially to product helices (Nunes-Düby et al., 1995). In this study we demonstrate a correlation between the isomeric structure of the naked Holliday junction substrate and the ability of λ Int to preferentially cleave, exchange and ligate the crossed pair of strands. We believe that these results have important implications not only for the geometry of the resolution reaction, but also for the complete recombination reaction. Furthermore, we have expanded upon an observation made by David Lilley's group (von Kitzing et al., 1990; Duckett et al., 1995): the stacking preference of certain branch point sequences that possess an inequality of purines and pyrimidines between opposing pairs of strands tends to maximize the number of purines in the crossed strands. Results Construction of central mobility Holliday junctions Spontaneous branch migration of a Holliday junction is an isoenergetic process that involves sequential dissociation and reassociation of hydrogen bonds between homologous (matched) base pairs. It is possible to block this process by the formation of even a single mismatched base pair (Quartin et al., 1989; Panyutin and Hsieh, 1993). A Holliday junction will consequently prefer to occupy regions of sequence homology, where full base pairing is permitted. It will refrain from entering regions of sequence heterology (where the DNA helices differ in sequence), since this would entail the formation of energetically unfavorable mismatched base pairs. This phenomenon has been widely exploited in the design of Holliday junction substrates with constrained mobility (de Massy et al., 1989; Dixon and Sadowski, 1994; Kho and Landy, 1994; Arciszewska et al., 1995; Lee and Jayaram, 1995; Nunes-Düby et al., 1995). Bilateral blocks to branch migration can be imposed by placing heterologous base pairs around the desired region of branch mobility. When the branch point is properly contained, the ‘barrier’ base pairs are fully matched; if the branch point moves beyond either one of these barriers, two of the four heterologous base pairs will become mismatched, depending upon whether the branch moves towards the C or C′ site. As stated above, this is an energetically unfavorable situation that is rapidly reversed. We had observed previously that immobile Holliday junctions with the branch point positioned 3 bp away from the top and bottom strand cleavage sites were exclusively resolved by λ Int at the top and bottom strands respectively. Monomobile Holliday junctions with access to both positions (3/4 and 4/5, Figure 2A and B) could be resolved in either direction (Nunes-Düby et al., 1995). However, the latter often displayed a bias of resolution that was sequence dependent. Since our model suggests an isomerization step at the center of the overlap region, we hypothesized that the resolution bias was linked to a preferred isomerization state of a monomobile Holliday junction. Figure 2.(A) Protocol to generate radiolabeled Holliday junction substrates. The substrates are assembled from four DNA duplexes generated by PCR, each containing a different pair of heterologous arm sequences: 3–4, 300 bp; 3–6, 216 bp; 5–4, 483 bp; 5–6, 399 bp. They each contain C and C′ λ Int core binding sites that surround a 7 bp overlap region. The 3 arm of the 3–6 duplex was labeled by performing the PCR reaction with a 5′-32P-end-labeled primer and a second unlabeled primer. These four duplexes are mixed in equimolar amounts, denatured and reannealed (see Materials and methods). Besides regenerating the original duplexes, two exchange forms of Holliday junctions are formed: types I and II. In this example only the type I Holliday junction is labeled. By convention, the ‘top strands’ are depicted by straight lines and the ‘bottom strands’ are depicted by wavy lines. The arrows indicate the positions of Int cleavage sites. Resolution at the top strands of the type I Holliday junction produces a 300 bp labeled duplex and resolution at the bottom strands produces a 216 bp labeled duplex. These products are fractionated by gel electrophoresis and are subsequently identified and analyzed by autoradiography (see Materials and methods). (B) Accessible branch point positions in the central mobility Holliday junctions. The base pairs in the overlap region are numbered from 1 to 7, starting immediately after the top strand cleavage sites and ending immediately before the bottom strand cleavage sites. The heterologous base pairs that impose the bilateral constraints to branch migration are depicted by bold slashes at positions 3 and 5. In the state shown to the left, the branch point sits at position 3/4 and in the state shown to the right, the branch point sits at position 4/5. Only the top strands-crossed isomer (TC isomer) is shown here; the bottom strands-crossed isomer has precisely the same junction mobility. (C) The logic used to construct central mobility Holliday junctions with opposite isomer preferences. Starting with a central mobility junction that has a unique isomer preference (TC isomer in this example) the central core of 6 bp is ‘excised’, rotated about 180° and replaced between the flanking arm sequences. This concept was not executed as diagramed, but was performed according to standard cloning procedures (see Materials and methods). It can be seen that the top strands have now become continuous with the bottom strands and vice versa. This construction will now prefer the BC isomer. Download figure Download PowerPoint In order to study such a correlation, a set of monomobile Holliday junctions was constructed (see Materials and methods; Figure 2A) where the branch point was confined to the center of the 7 bp overlap region with access to positions 3/4 and 4/5 (Figure 2B). These substrates needed to meet two requirements. First, each Holliday junction must have a unique and demonstrable isomerization state; it should predominantly assume a top strands-crossed isomer (TC isomer) or a bottom strands-crossed isomer (BC isomer). Second, each Holliday junction should permit analysis of the resolution bias (i.e. the relative proportion of top strand to bottom strand resolution). Our strategy to design Holliday junction substrates with opposite isomer preferences was based on the observation that the 4 bp that flank the branch point of an immobile Holliday junction are the exclusive determinants of which pair of strands are crossed and which pair of strands are continuous (Duckett et al., 1988). Since the branch point of a monomobile junction has access to two positions, 6 bp (3 bp from each duplex) now influence the global structure. We reasoned that an existing central mobility junction with an arbitrary but unique isomer preference could be used to rationally generate another central mobility junction with the opposite isomer preference. This could be accomplished by changing the orientation of the central core of 6 bp relative to the four distal arms of the Holliday junction (Figure 2C). As shown below, this proved to be a successful approach. A central mobility Holliday junction, HJ 10, of unknown stacking preference was selected (Figure 3A). Assuming that this construct preferred a unique isomer, another Holliday junction was constructed, HJ 11 (Figure 3A), in which the orientation of the base pairs that circumscribe the two possible branch point positions was altered according to the concept described above (Figure 2C). The sequences of these two Holliday junctions are identical except for the central 3 bp of the overlap region. Figure 3.Central mobility Holliday junctions. Holliday junctions are arranged into columns according to their isomer preference (TC or BC isomer), as determined by mobility shift assays, and are labeled according to their intrinsic bias [intrinsic top strand bias (IT), intrinsic bottom strand bias (IB) and no intrinsic bias (N)], as determined by resolution assays. The TC isomers are shown with their branch points at 3/4 and the BC isomers are shown with their branch points at 4/5, since these have been demonstrated to be the positions that favor top strand and bottom strand resolution respectively (Nunes-Düby et al., 1995). However, in every case the branch point has access to both positions 3/4 and 4/5. Heterologous base pairs that delimit the range of branch migration are in bold type; base pairs that affect the intrinsic bias are lower case. Top and bottom strand Int cleavage sites are indicated with downward or upward arrows respectively. ‘New’ constructs (A) and ‘old’ constructs (B) are listed separately. Download figure Download PowerPoint Isomer preference of central mobility junctions Gel electrophoretic methods have been extensively utilized to determine the structure of the Holliday junction (Cooper and Hagerman, 1987; Duckett et al., 1988, 1990). The validity of these approaches as a tool to infer the structure of the four-way junction has been independently confirmed by fluorescence resonance energy transfer studies (Murchie et al., 1989; Clegg et al., 1992, 1994), chemical probing studies (Chen et al., 1988; Churchill et al., 1988; Lu et al., 1989; Murchie et al., 1990, 1991), a molecular modeling exercise (von Kitzing et al., 1990) and other physical methods (Cooper and Hagerman, 1989). The experimental design of Duckett et al. (1988) employs immobile Holliday junctions with four arms of equal length (40 bp) that each have a unique restriction site located 12 bp from the branch point. All four arms are radioactively end-labeled, making it possible to identify the six permutations of doubly restricted Holliday junctions. These six species are electrophoresed through a high composition polyacrylamide gel in the presence or absence of divalent cations. The pattern of shifts that are observed reflect the disposition of the two long (unrestricted) arms in space. They determined that the rate of migration through the gel matrix is approximately proportional to the angle displayed by the two long arms. Since all of these species are the same size (i.e. they have the same number of base pairs), one is able to make qualitative comparisons between the relative mobilities of identical, or different, pairs of digests. When permuted sets of immobile Holliday junctions that prefer opposite isomers are electrophoresed under ionic conditions that either promote complete unstacking (2 mM EDTA) or efficient stacking (1 mM Mg2+) of the helical arms, characteristic patterns are obtained. Under the former condition the patterns of mobility shifts between the two sets are virtually identical, whereas in the latter condition the patterns of shifts between the two sets are dramatically different. To demonstrate which isomer prevailed (i.e. which pairs of arms were stacked on each other), our Holliday junctions were subjected to a gel permutation analysis modified from the method described above (Figure 4). Since it was essential for our Holliday junction substrates to have four arms of different lengths, in order to identify top and bottom strand resolution products (see below), mobility comparisons were made only between identically digested TC and BC isomers. Unique restriction enzyme cleavage sites were engineered into arms 4, 5 and 6. These cleavage sites are located 18–22 bp away from the branch point on their respective arms. Radioactively labeling arm 3 and performing sets of double digests allowed us to identify three of the six possible permuted species and to unambiguously differentiate their respective isomeric forms. Figure 4.Mobility shift assay to determine isomer preference. The Holliday junctions were uniquely labeled on the 3 arm with 32P (indicated by an asterisk). They were doubly restricted with either BamHI and BglII, BamHI and SalI or BglII and SalI to generate modified junctions with two long arms and two short arms. Pairs of these digested and undigested Holliday junctions were electrophoresed (see Materials and methods) either in 2 mM EDTA (A) or in 1 mM Mg2+ (B). The conditions used in (A) have been shown to promote complete unstacking of Holliday junctions into an extended structure, in which the four arms point towards the corners of a square (Duckett et al., 1990). The conditions used in (B) have been shown to promote complete stacking of Holliday junctions into one of two possible isomeric forms (Duckett et al., 1990): in the top strands-crossed isomer (HJ 10) (T) the 3 and 4 arms and the 5 and 6 arms are stacked on each other; in the bottom strands-crossed isomer (HJ 11) (B) the 3 and 6 arms and the 5 and 4 arms are stacked on each other. The stacked Holliday junction representations are shown in side view, in which it can be seen that the helical axes are related by an ∼60° angle (Duckett et al., 1988). The predicted conformations of the migrating species are symbolized by stick figures under each lane. The lengths of the arms and the positions of the unique restriction enzyme cleavage sites are indicated in (A). Download figure Download PowerPoint To establish which isomer the two Holliday junctions (HJ 10 and HJ 11) adopted, they were doubly digested with either BamHI and BglII, BamHI and SalI or BglII and SalI. Pairs of undigested and digested Holliday junctions were electrophoresed in 4% polyacrylamide containing either 2 mM EDTA or 1 mM Mg2+ (Figure 4). In 2 mM EDTA the mobilities were very similar between identical double digests of HJ 10 and HJ 11 (Figure 4A), suggesting that the molecules had similar conformations in this ionic environment. The variation in mobility between different types of digests is due to the different arm lengths that remain after restriction enzyme treatment. When the Holliday junctions were electrophoresed through the gels containing 1 mM Mg2+, the difference in mobilities between identical double digests of HJ 10 and HJ 11 was striking (Figure 4B). BamHI/BglII doubly-restricted HJ 10 had a retarded mobility compared with similarly digested HJ 11, as evidenced by an upward shift of the former and a downward shift of the latter species in the gel mobility shift assay. This suggests that 1 mM Mg2+ induced a (structural) transition that either facilitates or hampers migration through the polyacrylamide matrix respectively. The BglII/SalI-restricted HJ 10 and HJ 11 constructs showed the opposite trend. In this case, 1 mM Mg2+ ionic conditions induced a transition that allows the former species to migrate more rapidly than the latter. BamHI/SalI-restricted HJ 10 and HJ 11 constructs displayed virtually identical shifts, suggesting that these molecules exhibit similar characteristics under this ionic condition. The slight discrepancy in mobility between these species might be due to a minor variation in conformation. It should be noted that the BamHI/SalI doubly restricted Holliday junctions (with two opposing short arms) are less stable in 2 mM EDTA, where they tend to fall apart during the course of the electrophoretic run, as compared with 1 mM Mg2+. A small fraction of this species remains intact and can be seen on a darker exposure. All of the shifts can be explained in terms of HJ 10 existing as a TC isomer and HJ 11 existing as a BC isomer. Based on the models of Holliday junction migration through high composition gels (Cooper and Hagerman, 1987; Duckett et al., 1988), we interpret that the uncleaved long arms of BamHI/BglII-restricted HJ 10 (arms 3 and 6) describe an acute angled species, whereas the remaining long arms of similarly restricted HJ 11 describe an extended species. The migration pattern of the BglII/SalI doubly digested fragments is also consistent with the isomer prediction. This time we expect the faster moving HJ 10 digest to resemble a more linear, extended form and the retarded HJ 11 digest to resemble an acute angled conformation. The slight discrepancy in mobility between the uncleaved Holliday junctions is expected because of the slight pre-existing difference in the lengths of the arms. Indeed, this difference in mobility is consistent with the theory that the rate of migration is approximately proportional to the angle disposed between the two longest arms [in this case arms 4 (170 bp) and 5 (313 bp)], i.e. that the TC isomer will be retarded relative to the BC isomer, as observed. Resolution bias of central mobility Holliday junctions The Holliday junctions were designed with four arms of varying length so that placing a unique label on one of the arms (3 arm) would make it possible to distinguish between top strand and bottom strand resolution products, based on the relative sizes of the resulting radiolabeled duplexes (Figure 2A). Top strand resolution produces two linear duplexes: a 300 bp radiolabeled fragment and a 399 bp unlabeled fragment. Bottom strand resolution also produces two linear duplexes: a 216 bp radiolabeled fragment and a 483 bp unlabeled fragment. Following a resolution reaction, the DNA species can be separated by gel electrophoresis and subsequently identified and quantitated by phosphorimager analysis (see Materials and methods). All of the experiments reported here have been carried out with λ C65 Int, a C-terminal fragment that comprises the catalytic domain. This cloned peptide carries out topoisomerase function, forms a covalent complex with a suicide substrate and resolves Holliday junctions with the same efficiency as full-length λ Int (Pargellis et al., 1988; Tirumalai et al., 1996). In resolution reactions it has the advantage of giving linear reaction rates over a wider concentration of protein than intact λ Int and, in every instance tested, it gave identical results to intact λ Int (data not shown). Int (C65) resolution of the HJ 10 and HJ 11 Holliday junction constructs was assayed in 50 mM Na+, which has been previously shown to induce partial stacking (Duckett et al., 1990). HJ 11 displayed a weak top strand resolution bias (60–65%), whereas HJ 10 exhibited a 50% enhancement of this bias to give an almost exclusive top strand resolution (90–95%). It is therefore feasible to manufacture central mobility Holliday junctions with opposite isomers and, in the process, to dramatically alter their relative resolution bias. However, even the Holliday junction with the lower top strand bias still favors top strand resolution. We shall now show that the difference in bias is a consequence of the two different isomeric forms and that the intrinsic top strand bias is due to sequence effects at the site of strand cleavage. Intrinsic bias Suspecting that the DNA sequence at the sites of Int cleavage might significantly affect the efficiency of the cleavage and/or ligation reactions, it was noted that the top and bottom strand cleavage sites have a different base (Figure 3A). Specifically, at the position where Int cleaves and forms a transient covalent 3′-phosphotyrosine linkage there is a thymine at the top strand cleavage sites and an adenine at the bottom strand sites, i.e. adjacent to the overlap region on the left and right respectively. If this difference is the source of the intrinsic bias, then switching the arrangement of these two positions (i.e. placing A at the top strand cleavage sites and T at the bottom strand cleavage sites) would create a pair of Holliday junctions with an intrinsic bias to resolve their bottom strands. Accordingly, a second pair of Holliday junctions were constructed (HJ 6 and HJ 7) that were identical to the previous set except for having the sequences at positions −1 and 8 reversed (Figure 3A). HJ 6 resolved with a slight top strand bias (60–65%) in 50 mM Na+ and was shown to be a TC isomer in the gel permutation assay. HJ 7 resolved with a strong bottom strand bias (90–95%) and was shown to exist as a BC isomer (Figure 5). In other words, these two Holliday junctions show the same dramatic relative difference in resolution bias observed for HJ 10 and HJ 11, but now the intrinsic bias has been reversed to favor the bottom strands, as predicted. Figure 5.Quantitation of resolution assays for HJ 10, HJ 11, HJ 6 and HJ 7. The quantitation of Holliday junction resolution by gel electrophoresis and phosphorimager analysis is described (see Materials and methods; see also Figure 6). The resolution bias is the ratio of the percentage of top strand resolution to the percentage of bottom strand resolution and is plotted on a logarithmic scale. The constructs that display an intrinsic bias for top strand resolution (IT) (HJ 10 and HJ 11) versus an intrinsic bias for bottom strand resolution (IB) (HJ 6 and HJ 7) are bracketed in pairs. The isomeric preferences of the Holliday junctions are indicated by black bars for a TC isomer (HJ 10 and HJ 6) and by white bars for a BC isomer (HJ 11 and HJ 7). The results for three different concentrations of Int are shown. Download figure Download PowerPoint If the succ