ABSTRACT
Alignments of 105 site-specific recombinases belonging to the Int family of proteins identified extended areas of similarity and three types of structural differences. In addition to the previously recognized conservation of the tetrad R-H-R-Y, located in boxes I and II, several newly identified sequence patches include charged amino acids that are highly conserved and a specific pattern of buried residues contributing to the overall protein fold. With some notable exceptions, unconserved regions correspond to loops in the crystal structures of the catalytic domains of [lambda] Int (Int c170) and HP1 Int (HPC) and of the recombinases XerD and Cre. Two structured regions also harbor some pronounced differences. The first comprises [beta]-sheets 4 and 5, [alpha]-helix D and the adjacent loop connecting it to [alpha]-helix E: two Ints of phages infecting thermophilic bacteria are missing this region altogether; the crystal structures of HPC, XerD and Cre reveal a lack of [beta]-sheets 4 and 5; Cre displays two additional [beta]-sheets following [alpha]-helix D; five recombinases carry large insertions. The second involves the catalytic tyrosine and is seen in a comparison of the four crystal structures. The yeast recombinases can theoretically be fitted to the Int fold, but the overall differences, involving changes in spacing as well as in motif structure, are more substantial than seen in most other proteins. The phenotypes of mutations compiled from several proteins are correlated with the available structural information and structure-function relationships are discussed. In addition, a few prokaryotic and eukaryotic enzymes with partial homology with the Int family of recombinases may be distantly related, either through divergent or convergent evolution. These include a restriction enzyme and a subgroup of eukaryotic RNA helicases (D-E-A-D proteins).
The crystal structure of the minimal catalytically active C-terminal domain of Int, called [lambda] Int c170 (residues 175-356; 1), has been determined at 1.9 Å resolution (2). More recently, crystal structures of the C-terminal domain of the Haemophilus influenzae phage integrase HP1 (HPC, residues 165-337) and of the Escherichia coli resolvase XerD have been determined at 2.7 and 2.2 Å resolution respectively (3,4). In addition, the structure of the Cre recombinase complexed to DNA was most recently reported at 2.4 Å resolution (5). These four structures allow a more informed alignment of the ever growing number of `Int family' site-specific recombinases than was previously possible (6-11). As of September 1997, >130 complete sequences of proteins have been assigned to this family from Archaea, Eubacteria and their phages, from a mitochondrion and from yeast. Among these, 105 proteins are distinct and have been well characterized or identified as belonging to a well-studied subgroup [listed in Table 1 (12-47) and 2 (8,48-89)].
Functions of site-specific recombinases include integrative and excisive recombination of viral and plasmid DNA into and out of the host chromosome, conjugative transposition, resolution of catenated DNA circles, regulation of plasmid copy number, DNA excision to control gene expression for nitrogen fixation in Anabaena and DNA inversions controlling expression of cell surface proteins or DNA replication (83,90-93). Alignment of this family of protein sequences may facilitate a better understanding of the structure-function relationship of these proteins through identification of residues and secondary structures implicated in catalysis, specific and non-specific DNA binding, protein-protein interactions and the overall protein fold.
These site-specific recombinases utilize a topoisomerase I-like mechanism, cleaving and rejoining one strand of DNA per protomer (94). A complete recombination event therefore requires at least four molecules of the recombinase, two on each DNA recombination partner (95-97). DNA strand exchange is conservative in two ways: there are no deletions or additions of nucleotides at the site of exchange and there is no need for high energy cofactors. A transient 3'-phosphotyrosine linkage between protein and DNA conserves the energy of the cleaved phosphodiester bond. The covalent protein-DNA intermediate is resolved by nucleophilic attack on the phosphotyrosine bond by the 5'-terminal hydroxyl of the invading strand. Proteolysis of [lambda] Int under native conditions yields a C-terminal fragment, [lambda] Int c170 (residues 170-356), which was subsequently cloned and expressed in E.coli. [lambda] Int c170 contains all the catalytic residues needed for type I topoisomerase-like cleavage and ligation of DNA (1), including the two conserved sequence boxes that are diagnostic for Int family recombinases (6).
Our analysis of the catalytic domains from Int family recombinases benefits from the inclusion of many newly identified sequences and from the recent crystal structures of four family members. We explore the similarities and differences of all members of the Int family of site-specific recombinases aligned by automated procedures (98), combined with manual editing. These new alignments identify several new sequence motifs that relate to the structures and biological activities of these recombinases. We also compile the mutational studies of a subgroup of Int family recombinases, in order to correlate the phenotypes of the mutants with the overall tertiary fold and/or the structure and function of the catalytic pocket. Furthermore, we extend our comparisons to more distantly related proteins.
The primary sequences of 111 site-specific recombinases (listed with references in Tables 1 and 2) were collected by multiple searches of the databanks (GenBank, Swissprot, EMBL and Pir). The following keywords were used: Int, integrase, recombinase, Int family, transposase, resolvase, invertase, excisionase, Xis, Xer, Fim, Flp and shufflon. Individual searches returned two to ~40 different sequences, in addition to duplicates and false returns. This variability probably results from differences in databank entries by different authors (including DNA or amino acid sequences, descriptions and keywords) and from the use of the same keywords for different families of proteins. In addition, blast searches were performed with sequence strings carrying the conserved `box I' (9) and/or `box II' residues (6). Interestingly, ~24 sequences were not recovered by blast searches (see also 11). These searches were hampered by the low number of residues (three) that are 100% conserved in all members of this family of recombinases. A number of recombinases that likely belong to this family could not be included due to lack of or incomplete sequencing data (99,100; W.B.White, unpublished results, accession no. L39071).
Table 1.
x) vap, virulence associated protein.
Table 2.
The 105 protein sequences analyzed here were compiled from 111 citations with 99 prokaryotic (including Archaea and one mitochondrial protein) and six yeast recombinases (Tables 1 and 2). Approximately 24 ORFs from different organisms assigned to `tyrosine recombinases' without biochemical characterization were not included (see Materials and Methods). The alignment in Figure 1 is derived from the 99 unique prokaryotic proteins, although 19 of these have not been included in this figure for reasons of space and clarity (listed in Materials and Methods). These comprise 11 sequences with >94% identity to the catalytic domains of other family members. Furthermore, eight sequences retrieved after 1 September 1997 are not shown in Figure 1 but are part of the calculation used for establishing the consensus sequence in Figure 2; these have similarity scores >90% to their respective homologs. As a result, 94 distinct recombinases (88 prokaryotic and six eukaryotic) are analyzed here (Fig. 2), of which 80 prokaryotic sequences are aligned in Figure 1.
The catalytic domain of the Int family of recombinases spans ~180 amino acids. The shortest members belonging to this protein family, aligned to [lambda] Int, start very close to the protease-accessible A170 of [lambda] Int. The N-terminal methionines of pCL1, FimE, pDU1, FimB and MrpI recombinases correspond to [lambda] Int positions 176, 174, 169, 168 and 157 respectively (Fig. 1A). Almost all the other members of the Int family carry one or more prolines at positions equivalent to or neighboring A170. Catalytic domain fragments identified in HP1, Cre and Flp by partial proteolysis start at residues K165, R119 and S129, equivalent to [lambda] Int coordinates 171, 158 and 156 respectively (3,121,122). In the crystal structures of XerD and Cre an unfolded linker separates the distinct N-terminal domain from the C-terminal catalytic domain (4,5). The first [alpha]-helix of their catalytic domains, labeled E in XerD and F in Cre, align with [alpha]-helix A of [lambda] Int c170.
All proteins harbor two regions of marked sequence similarity, here called `box I' and `box II', originally identified from alignment of only eight recombinases, seven derived from bacteriophages [lambda], [Phi]80, P1, P2, P4, P22 and 186 and the yeast protein Flp (6). Boxes I and II were first limited to 13 residues from M203 to D215 and to 37-39 residues from H308 to D344, respectively. These authors identified three residues in box II that were 100% conserved, the triad H-R-Y, which includes the active site tyrosine (7). With alignment of 22 prokaryotic and six yeast recombinases the box I sequence was expanded to 21 residues, ending with D223, and a fourth absolutely conserved residue, R212, was identified (9). The first of two conserved regions among the six Flp proteins of Saccharomyces and Zygosaccharomyces is homologous to box I, shortened left and right by four and three residues (8). The second conserved region comprises parts of [alpha]-helix F (with the conserved H and R) and the preceding loop (Flp sequence IFAIKNGPKSHIGRHLMTS), i.e. it only partially overlaps with the box 2 sequence shown in Figure 2. The conserved tetrad R-H-R-Y has been established by mutational analyses as the hallmark for the Int family of recombinases (see below, Table 4). Two more recent analyses, limited to box I (box A) and/or box II (Box B/C) of 58 and 80 members respectively confirmed the original alignment, but distinguished the eukaryotic from the prokaryotic sequences (10,11).
While scanning for the presence of the R-H-R-Y signature we find that the two arginines and the tyrosine are indeed invariant in the larger group of Int family recombinases assembled here. However, eight recombinases show a substitution of the highly conserved histidine by either an arginine (actinophage Rp3 and pSAM2), a lysine (Sulfolobus phage Ssv1), an asparagine (phage [Phi]CTX and Baculovirus factor Vlf-1) or a tyrosine (Slp1 element, cyanobacterial XisC and XisA). In support of a less stringent requirement for a histidine at that site is the observation that two mutants, His289Tyr of Cre and His305Gln of Flp (see Table 4), retain at least partial recombination activity (123-125).
For the purpose of presenting the alignments each recombinase was partitioned into three segments comprising the two conserved regions, box I (A202-G225 in [lambda] Int) and box II (T306-D344 in [lambda] Int) and the interval between them. The junctions between these segments were chosen within regions that are devoid of secondary structure in crystal structures of [lambda] Int c170, HPC, XerD and Cre. The junctions are located at Q233 (in a [beta]-turn between [beta]-sheets 2 and 3) and G297 (in the loop between [alpha]-helices E and F) of [lambda] Int. The first segment spans from V175 to Q233 and contains box I (Fig. 1A). The middle segment spans from S234 to G297 (Fig. 1B) and the last segment, including box II, spans from L298 through the C-terminal Q337 of HP1 (Fig. 1C). The lengths of these segments differ among Int family members because of insertions and deletions located between the elements of regular secondary structure.
The high sequence conservation of boxes I and II, including the triad R-H-R, is reflected in the conserved secondary structure of [lambda] Int c170, HPC, XerD and Cre (2-5). In each of these proteins the R-H-R residues form a cluster on the protein surface, located at the center of the DNA interaction surface in the Cre-DNA complex. R212 (HPC R207, XerD R148 and Cre R173) lies on the short loop between [alpha]-helices B and C ([alpha]2 and [alpha]3 in HPC, [alpha]F and [alpha]G in XerD and [alpha]G and [alpha]H in Cre); H308 (H280, H244 and H289) and R311 (R283, R247 and R292) are located at the N-terminal end of [alpha]-helix F ([alpha]6 in HPC, [alpha]L in XerD and [alpha]K in Cre). [alpha]-Helices B and C with the conserved R212 constitute box I and form the very core of the protein, with a large number of buried residues (Fig. 1A). In addition, these helices harbor six highly conserved polar or acidic amino acids (highlighted in green and magenta respectively) that form one flank of the catalytic pocket. The function of these conserved residues is not yet known, although most mutations of D215 in P2 Int and in Flp decrease DNA binding and compromise topoisomerase and recombination functions (Table 4). The conservation of box I is striking in prokaryotic recombinases (Fig. 1A) and it extends with some variations to eukaryotic recombinases (Fig. 2).
Box II, which includes three of four residues of the R-H-R-Y motif, is also relatively strongly conserved among the prokaryotic recombinases (Fig. 1C), but less so between prokaryotic and eukaryotic proteins (Fig. 2). Among prokaryotic recombinases residues in [alpha]-helices F and G ([alpha]6 and [alpha]7 in HPC, [alpha]L and [alpha]M in XerD and [alpha]K and [alpha]L in Cre) are particularly well conserved, as is the separation between residues corresponding to H308 and Y342 of Int. The shortest separation between these catalytically important residues is that of phage 21, with 31 amino acids, the bulk (81 recombinases) carries 33-35 amino acids, five have 36 amino acids and the longest is that of MV4 Int, with 37 amino acids. The yeast recombinases, in comparison, have a longer segment between the catalytic histidine and tyrosine ranging from 37 (Flp) to 40 residues (see below). Whereas the active site tyrosine is absolutely conserved, the surrounding residues are rather divergent, allowing for quite different secondary structures, as discussed below.
The crystal structure of the [lambda] Int catalytic domain revealed a pattern of conserved hydrophobic residues that form the core of the globular structure (2; Fig. 3). These include: L180, Y185, Ile188, Tyr189, Met203, Leu205, Val207, Val208, Leu216, Met219, Ile224, Leu229, Val231, Ile242, Pro243, Leu251, Met255, Ile271, Ile272, Leu280, Val285, Phe289 and Leu330. Amino acid substitutions at the positions of the underlined residues (above) cause defects in recombination to varying extents (see Table 4). The high degree of conservation and clustering of hydrophobic residues is evident from the alignments. As supported by the available crystal structures (2-5), this conservation of core residues suggests that all members of the integrase family adopt similar folds for the region spanning box I, the interval region and box II (see the score for per cent hydrophobicity in Fig. 2). From the alignment of the 88 distinct prokaryotic recombinases (with <94% identity), per cent identity and per cent similarity are reported at positions where similarity (belonging to the same exchange group; 104) is at least 50%. A consensus sequence of the prokaryotic recombinases, derived from residues with similarity scores >50% and/or identity scores >31%, is shown in Figure 2.
The usefulness of primary sequence alignments and predicted secondary and tertiary structure comparisons lies not only in identification of similarities important for similar functions of closely related proteins, but also in recognition of their differences. The latter may lead to an understanding of functional variations affecting both specificity and efficiency of the reactions in question. We consider three types of structural differences observed among family members that may also have functional significance. These are revealed by differences in the crystal structures of [lambda] Int c170, HPC, XerD and Cre and they are evident from the aligned sequences, especially from the presence of large insertions or deletions. These differences involve: (i) the least conserved `interval' sequence located between boxes I and II, which lies on a surface of the protein away from the DNA interaction interface; (ii) the secondary structures of box II; (iii) the sequence motifs and their spacing in eukaryotic versus prokaryotic recombinases (Fig. 2). These three types of differences will now be discussed in more detail.
First, the most striking differences in primary sequence and corresponding higher order structure are located between the conserved [beta]-sheet 3, following box I, and [alpha]-helix E, preceding box II (Fig. 1B; note that our alignment of HP1 with [lambda] Int differs from that published by Hickman et al.; 3). In [lambda] Int c170 this region contains [beta]-sheets 4 and 5, [alpha]-helix D and a compound loop; in HPC only [alpha]-helix 4(D) and a small loop are present; in Xer D there are two [alpha]-helices and a compound loop; in Cre the longer [alpha]-helix I (equivalent to [alpha]-helix D) is followed by a shorter compound loop and two small [beta]-sheets. Interestingly, these surface differences among the crystallized proteins do not significantly alter the overall fold of the protein cores, which can easily be superimposed on each other. It appears that most recombinases resemble in size and primary structure one of the four proteins that have been crystallized. The two integrases derived from organisms that thrive at high temperatures, Sulfolobus phage Ssv1 and SFi21 phage of Streptococcus thermophilus, lack most of this region, although they both carry the patch III sequence preceding [alpha]-helix E. The Ints of pSE211 and pSE101 carry two inserts, the first is large (58 and 66 amino acids), just upstream of [beta]-sheet 4 and rich in proline/glycine and the second is small, following or extending [alpha]-helix D. All integron Ints also carry large inserts, located on both sides of patch III. The significance of these changes are not yet known, although their surface location away from the active site speaks against direct involvement in the cleavage and ligation functions.
Second, the structures determined from crystals of [lambda] Int c170, HPC, XerD and Cre reveal fundamental differences in the region of the catalytically active tyrosine. This is important because of the two distinctive modes of DNA cleavage, in cis or in trans, observed in different systems and under different conditions. cis-cleavage occurs when the tyrosine nucleophile attacks the DNA site bound by the same protomer. trans-cleavage is accomplished when the tyrosine of one protomer cleaves a DNA strand that is bound and activated by the R-H-R triad of a neighboring protomer (128). Some in vitro complementation tests suggested that Cre of phage P1 might cleave in trans (129). However, the structure of the co-crystal clearly shows the tyrosine in cis mode (5). Although [lambda] Int has been shown to cleave in cis during Holliday junction resolution and suicide substrate cleavage, trans cleavage has also been suggested in a different experimental context (130,131). Because Y342 of [lambda] Int is located next to a flexible loop, it could be delivered into the catalytic Arg-His-Arg cleft in either a cis or a trans configuration (2). When the loop bends backward toward the protein core the catalytic tyrosine is very close to the highly conserved triad Arg-His-Arg of the same protomer (cleavage in cis), whereas the tyrosine is located 17 Å removed from each of the two conserved arginines and 23 Å from the histidine of the same molecule when the loop is stretched out. In this more extended conformation the active site tyrosine might reach into the catalytic pocket of another protomer bound to a different DNA site, leading to cleavage in trans.
On the other hand, Y315 of HP1 Int, Y279 of XerD and Y324 of Cre all sit in an [alpha]-helix with a relatively fixed position. The tyrosine points toward the defined active site cleft of the same protomer in HP1 and Cre, consistent with cleavage in cis. In the XerD crystal the tyrosine appears to be buried, which suggests an inactive conformation in the absence of the partner recombinase XerC and DNA. When it cleaves, XerD, like its partner recombinase XerC, has been shown to act in cis (49,132). It is interesting that XerD-mediated cleavage depends on the structure of its substrate: psi sites are readily cleaved, whereas cer sites are not, despite stable complex formation with either substrate (133).
Variations in the sequence and spacing of conserved motifs of the eukaryotic recombinases, in comparison with the prokaryotic recombinases, constitute the third type of changes mentioned above. In theory the sequences of the eukaryotic recombinases can be threaded into the tertiary fold of [lambda] Int or a related protein of known structure, but several unique features of the eukaryotic sequences are suggestive of a significantly different structure (Fig. 2). An attempt to map the six eukaryotic sequences onto an evolutionary tree of prokaryotic sequences was not successful (11). Nonetheless, a recent theoretical model of the yeast Flp protein has a fold that is generally consistent with existing structures of prokaryotic recombinases (134,135). The best fit of this model structure with the actual crystal structures was found within the region of box I and beyond to encompass [beta]-sheet 3.
In the Flp-type recombinases differences in the spacing between conserved motifs, one to the left of box I and the other within box II, hint at a functional difference in comparison with the prokaryotic recombinases. The Flp recombinase cleaves its target sites in trans and this mode of function might require an increase in the length of the segment corresponding to box II, as was proposed by Blakely and Sherratt (10). This difference in spacing is most evident when aligning Int G332 with Flp G228. Whereas the distance between this glycine (at the end of [alpha]-helix G) and the conserved tyrosine is found to be nine or 10 residues in all prokaryotic proteins, it is longer in all yeast proteins, varying between 14 and 17 residues. Interestingly, there is a protease-sensitive site in Flp between R340 and the active site Y343, supporting the notion of an extended easily accessible loop (122). This is not unlike the protease-sensitive site observed in [lambda] Int within the disordered loop that spans this region (1).
The yeast recombinases also display some critical sequence changes in comparison with prokaryotic proteins. Two motif changes lie within the most conserved regions, box I (at coordinates 209-212) and box II (at coordinates 330-333): the prokaryotic box I motif `TGXR' appears as `NCCR' and the prokaryotic box II motif `LLGH' or `LLGW' is shifted and reads `[LVSP]-[YFLV]-GNW'. Whereas all reported mutations of the box I sequence in Flp cause a recombination defect, several box II mutants retain full (N329H) or partial activity (N329D) (136,137). It is possible that the tryphophan following N329 in all yeast recombinases is the functional equivalent of W315 of Cre, as was first suggested by Guo et al. (5). Additional differences are prominent within the newly identified patches that show sequence conservation in prokaryotic proteins. The yeast recombinases only share the right half ofprokaryotic patch I (EEV - - LL), with the slightly modified consensus ESI - - FV. Within patch II only the `TKT' of the eukaryotic sequences aligns well with prokaryotic sequences. The yeast proteins have three strings in tandem that poorly fit the patch III motif (HIYFFS<5>DPLVYLD<5>EPYPKS); however, only the third string fits the location of this sequence patch in prokaryotic proteins, while the first lies in patch II, overlapping with [beta]-sheet 3.
Whereas the loss of function associated with mutating the catalytic tyrosine has often been used to establish Int family membership, a more detailed analysis of point mutations has been performed with only a few proteins, including the Ints of phages [lambda] (7,126, 127,130,131,138-140) and P2 (J.Eriksson and E.Haggård, personal communication), Cre of P1 (9,123,141,142), Flp and the related yeast recombinase R (124,125,136,137,143-150; Table 4). Mutations are labeled by residue changes and numbers referring to the recombinase that was mutated. For the purposes of locating the mutant positions in the alignment of Figure 1 the analogous positions of the [lambda] Int sequence and their respective secondary structures are given as coordinates (Table 4, first 3 columns). In addition to point mutations, one C-terminal deletion and three small insertion mutations were included in the compilation. A two residue insertion in Cre, located on the loop preceding [beta]-sheet 1, had wild-type activity. Two four residue insertions in Flp, one lining up with [beta]-sheet 1, the other with [alpha]-helix E, abolish DNA binding as well as recombination.
Larger insertions and deletions of XerD have been analyzed in great detail and are presented elsewhere (150,;Sherratt and Hayes, personal communication). The only truncated recombinase that retains some activity is [lambda] Int W350ter; it is defective for recombination, but resolves Holliday junctions and has increased topoisomerase activity (150). This is a surprising result, because the truncation removes [beta]-sheet 7, which in the Int c170 crystal structure is firmly anchored to the rest of the protein (150). In crystal structures of HPC and Cre two C-terminal [alpha]-helices of adjacent protomers form an extensive dimer interface (150,150). Although [lambda] Int lacks a segment corresponding to these C-terminal helices, adjacent parts of its structure could also participate in protein-protein interactions.
Permissive sequence changes include a set of four mutations of [lambda] Int, located on the outer surfaces of [alpha]-helices E and F, that (in conjunction with a fifth change, N99D) cause a switch of binding specificity from the [lambda]-type to the HK022-type recognition sequence for core DNA of attachment sites (150). Another mutation on the surface of [alpha]-helix E (R293Q) is deficient in core binding and isolated cleavage reactions, but retains some Holliday junction resolution and in vivo recombination activity (150). Most other permissive point mutations involve substitutions of residues with similar character (same exchange group) or residues located at positions away from the active site or within a connecting loop. However, one well-tolerated mutation was quite unexpected and surprising: The highly conserved acidic residue close to the first `trademark' arginine, Asp194 in Flp, could be mutated to a tyrosine with impunity, whereas a change to a glycine or asparagine was detrimental (150; H. Friesen, PhD thesis, University of Toronto, Canada, 1992). In [lambda] Int this Asp215 forms a water-mediated interaction with Arg212 (150).
Mutations with a defective phenotype fall into four categories. (i) Mutations that change the catalytic tyrosine prevent cleavage; in Flp these recombination-deficient mutants have been shown to catalyze ligation in cis on nicked `activated' substrates carrying a phosphotyrosine bond (150, 150). (ii) Mutations that affect the hydrophobic and other core residues disturb the tertiary fold (in [lambda] Int M220K, 1242N, T270I, S274F, P304L and P305L; 150). (iii) Mutations that alter the H-R-H triad fall into two subgroups: whereas some of these mutants with a change from one exchange group to another (150) are deficient for all functions, the `step-arrest' mutants of Flp, including Arg191Lys, His305Leu/Pro and Arg308Lys, can bind to the target site and promote cleavage, but are ligation deficient (150,150,150,150,150,150,150). (iv) There are some mutants for which the defect is not readily understood, since they do not alter residues involved with catalysis and would not be `predicted' to have a large structural effect: they include Ala199Val in P2 Int, Met290Ile in [lambda] Int and Gly288Val in Cre.
We noted above that the two conserved histidines in box II (H308 and H333 in [lambda] Int) are symmetrically positioned on either side of the two conserved arginines (R212 and R311). It is interesting that the two recombinases that substitute H333 with a residue other than tryptophan, namely arginine in Ssv1 and aspartate in pC2A, both belong to Archaea and carry a number of unique substitutions at other conserved positions, particularly in the box II region, e.g. the first conserved histidine (H308) is replaced by a lysine in Ssv1, perhaps as a compensatory change. The Ssv1 sequence is more divergent from those of other Int family recombinases throughout its length and it maps most distantly on an evolutionary tree (7).
A few protein sequences in the databanks that were ascribed to the Int family of recombinases could not be fitted into our alignments (Table 3). These include the Ints of corynephage AAU2, [Phi]AR29 and frog virus FV3 (112-114), as well as the immunoglobulin [kappa]J recombination signal protein (RBP-J[kappa]) from human, mouse, Xenopus, Drosophila and yeast (117-119). The latter proteins have the triad R-H-Y (reversed H-R-Y motif) with the correct spacing near their C-terminus, but they lack the internal arginine and other conserved sequence patches. They were recently identified as transcription factors (152). Although the eukaryotic RAG I proteins show some homology with Fim B/E, with good alignment of the conserved R-H-R, the best fit is with non-conserved residues of the Int family recombinases (115,116). In addition, RAG I proteins have no correctly spaced tyrosine in the region equivalent to box II. Instead, a serine aligns with the tyrosine of Flp, the significance of which is questionable.
We have included the very late transcription factor Vlf-1 of baculovirus Autographa californica in our alignment, although no recombination function is known for this protein (47,153). Vlf-1 transactivates the polyhedrin gene, polh, required for occluded virus formation (polyhedrosis). The fit with the Int family of recombinases, first recognized by McLachlin and Miller (47), is exceptionally good, suggesting a secondary recombination function for Vlf-1. This is very exciting because the insect baculovirus is evolutionarily very distant from the bacteriophage. It is noteworthy that another member of the Int family, the resD protein of the Escherichia coli miniF plasmid, also has two independent functions, one as a repressor of transcription in the ori-1 region and the other as a site-specific resolvase (154).
Some prokaryotic proteins, an IS1 transposase (InsAB') and the restriction enzyme EcoRII, may be distantly related to the Int family of recombinases, although not necessarily through evolutionary divergence from a common ancestor (Table 3). Neither show a good fit for box I, but both carry some or all of the conserved box II residues of [lambda] Int (Fig. 2). The spacing between H308 and Y342 is shorter than that observed in any of the Int family members proper, namely 30 and 26 amino acids in the C-termini of InsAB' and EcoRII respectively (155-157). In contrast, the Int family spacing varies between 33 and 37 in prokaryotic and between 37 and 40 in eukaryotic recombinases. Phage 21 Int is the single exception, with the shortest box II sequence of 31 amino acids. InsAB' also carries the internal motif of box II, VIGH, separated from the tyrosine by six amino acids (compared with eight or nine in prokaryotic Int family members). Interestingly, mutational analysis of the H-R-Y triad in InsAB' revealed that its transposase activity depends on all three conserved residues (155). Similarly, a Y308F mutation in EcoRII abolishes its cleavage function (157). EcoRII belongs to the type IIe enzymes that require two recognition sites for their function (158). It may be noteworthy that another type IIe enzyme, the endonuclease NaeI, carrying a single point mutation (L43K), displayed sequence-specific DNA topoisomerase and recombinase activities (159). However, the NaeI sequence could not be aligned with sequences of Int family members.
The `D-E-A-D box' subfamily of eukaryotic RNA helicases (four members are shown as representatives for this large family; Table 3 and Fig. 2) show substantial overall similarities to the Int family recombinases, especially in boxes I and II, with absolute conservation of the two arginines (R212 and R311 in [lambda] Int). A particularly striking alignment with the baculovirus transcription factor Vlf-1 had previously been shown by McLachlin and Miller (47). However, even within boxes I and II there are some critical substitutions of highly conserved amino acids in individual members of this helicase subfamily (Fig. 2). In other words, the most conserved residues in members of the Int family are not particularly conserved in members of this helicase family (except for the two Arg).
The sequence alignment presented here is based upon the crystal structures of four Int family members. In conjunction with biochemical analyses of mutated proteins, they allow us to generalize the involvement of specific residues and/or certain regions of these recombinases in particular functions. These include catalysis, DNA binding, binding specificity and protein-protein interactions to ensure correct multimerization in an active recombination complex. Strong protein-protein interfaces have been identified at the extreme C-termini of HPC, XerD and Cre. Catalytic activity is likely to depend not only on the presence of the `signature' tetrad R-H-R-Y, but in addition on the following conserved residues that appear to comprise the catalytic pocket: D215, which forms a water bridge with R212; K235, that, in Cre, is shown to make a direct contact with DNA adjacent to the site of DNA nicking (5); H333 (W313 in Cre). In the structures of HPC and XerD two additional highly conserved histidines, not present in [lambda] Int and Cre, are located near the arginine and tyrosine of the box II motif, within the enzyme active site. These are also present in Flp; mutations at either of these two positions render Flp inactive.
Although [lambda] Int c170 has catalytic activity, it does not bind tightly to the core sequence of the phage attachment site by itself. A critical component of the core binding domain resides in the region immediately N-terminal of residue 170 (1). Similarly, some core DNA binding properties have been assigned to the analogous N-terminal domains of XerD and Cre (4,5). However, the catalytic domain undoubtedly contributes to DNA binding and/or binding specificity. The five shortest proteins, FimB, FimE, MrpI, pCL1 and pDU1, which lack upstream (N-terminal) and downstream (C-terminal) sequences, nevertheless recognize and bind DNA to carry out their respective recombination functions. Two other recombinases with very short upstream N-terminal sequences, ResD of F factor and TnpA of Weeksella, carry a small insert between patch III and [alpha]-helix E, similar to Cre (Fig. 1B). The DNA-Cre co-crystal reveals two [beta]-sheets in this region that make extensive specific DNA contacts at the periphery of the complex (5).
Three lines of evidence point to [alpha]-helix E as a site of sequence-specific DNA recognition within the catalytic domain: (i) R259 of Cre, located at the beginning of [alpha]-helix K (equivalent to G283 in [alpha]-helix E of [lambda] Int) forms two specific hydrogen bond interactions with a guanine at the center of the core recognition sequences of lox sites, seven bases removed from the cleavage sites (5); (ii) three of the five `core specificity' mutants of [lambda] Int, responsible for a switch of DNA recognition from a [lambda]-type to an HK022-type sequence, are located at the beginning of [alpha]-helix E and these three surface residues, S282P, G283K and R287K, are in positions overlapping the DNA binding interface of the Cre protein; (iii) the exact same positions of the equivalent [alpha]-helix J in XerC and XerD have been implicated in their respective binding specificities (4). These authors pointed out a structural similarity of this region to the DNA binding domain of E.coli CAP protein. In addition to sequence homology, there is a tertiary structure similarity between the helix-turn-helix motif of CAP and two separated helices of the crystallized recombinases, e.g. [alpha]-helix G and [alpha]-helix J in XerD ([alpha]-helix C and [alpha]-helix E in [lambda] Int). A helix-turn-helix fold comprised of two non-adjacent helices has also been reported for endonuclease FokI (160). It is notable that [alpha]-helix E is exceptionally rich in basic residues, although their positions are not strictly conserved. Positively charged residues occur preferentially at the six positions on the hydrophilic surface of this amphipathic helix (i.e. 26, 43, 53, 36, 24 and 37% at positions 283, 287, 290, 291, 294 and 295 respectively).
In summary, several new sequence motifs have been identified in the catalytic domains of Int family site-specific DNA recombinases. The crystal structures of four Int family members show that these conserved patches include groups of buried residues, which define the common fold of these proteins and residues clustered in and around the enzyme active site. Pronounced differences in the sequences and structures are present in the C-terminal region, forming subunit interactions during synapsis, and in segments flanking the catalytic tyrosine nucleophile. Differences in the position of the catalytic tyrosine and the surrounding secondary structure may underlie the mechanistic differences in proteins that cleave DNA in cis or in trans. An additional complexity is present in the N-terminal segment of some Int family recombinases, in a region not covered by our sequence alignments. Some Int family members have a second N-terminal DNA binding domain that binds to specific sites flanking the site of DNA cleavage and thereby assists in DNA strand exchange. It is not known whether this N-terminal DNA binding domain directly contacts the C-terminal catalytic domain, but we might expect such an interacting surface to be located on the unconserved face of the catalytic domain, away from the active site. The sequence alignments of the catalytic domains presented here will help guide and interpret future biochemical analyses of the Int family of recombinases.
We thank Drs D.J.Sherratt, D.B.Wigley, G.D.van Dyne, M.Jayaram, J.W.Golden, L.Dijkhuizen, P.Roy, J.Eriksson and E.Haggård for communicating data before publication. We thank Jeffrey C.Liu for computer programming and Joan Boyles for help with preparation of the manuscript. This work was supported by NIST grant 60NANB5D0009 (S.E.N.-D.), a Howard Hughes Medical Institute Predoctoral Fellowship (H.J.K.), the Lucille P.Markey Charitable Trust (T.E.E.) and NIH grants AI13544 and GM33928 (A.L.).
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Results And Discussion
The basic blueprint of Int family recombinases
Additional similarities among Int family members
Major differences among Int family members
Mutational analysis of Int family recombinases
Related proteins
Structure-function relationships
Acknowledgements
References
+) Very late transcription factor of the eukaryotic baculovirus A.californica, nuclear polyhedrosis virus (AcMNPV), activating the polh gene involved in formation of polyhedral occlusion bodies; no recombination functions are known for Vlf-1. The following phages share >98% identity and appear as a single entry in the alignments (Figs 1-1):
*1 phage [lambda] also represents phage 434;
*2 phage SF6 also represents phage YfdB;
*3 phage P22 also represents phage Dlp12
;*4 phage HP1 also represents phage S2;
*5 phage [Phi]LC3 also represents phages Tuc2009, BK5-T and phi r1t.
The database sources for accession nos are SwissProt (starting with a P), GenBank, EMBL and Pir. When multiple cross-references were available the SwissProt no. was preferentially entered. The NCBI Id no. refers to NID (PID in the case of multigene entries). Among multiple databank entries the highest NCBI sequence Id nos were chosen, as they are more likely to include the most recent updates.
CLEAR=ALL>
*2 These open reading frames (ORFs) of putative Int family members were recovered too late to be incorporated into the alignment shown in Figure 1. The translated sequences fit the consensus and show a particularly high degree of similarity with the respective groups of proteins they have been associated with in this Table (see also Materials and Methods). For H.pylori Xer proteins subfamily assignment is hypothetical.
*3 The newly identified Xer of M.leprae has strongest similarity to `XerD' of M.tuberculosis (88% identity, 93% similarity). Assignment to the XerC or XerD subfamily is as yet hypothetical.
*4 Share >94% identity, represented by homologs of E.coli.
*5 Share 98% identity, represented by resD of F factor as a single entry in sequence alignments.
*6 For a list of different organisms see SwissProt file: IntR_ecoli/P09999.
*7 This recombinase is only active when the internal termination codon is removed.
*8 NBU, non-replicative bacteroides unit.
*9 Carries one mutation: N308D.
*10 Shares 86% identity and 92% similarity with the other E.coli shufflons.
*11 Although recovered from different (though closely related) organisms, these proteins are identical within the catalytic domain.
*12 A tnpB homolog (S.aureus) has also been reported by Chikramane and Dubin (unpublished results), with NCBI Id no. 586103, accession no. P37375.
*13 XisC and XisA are necessary for site-specific excision of the 10.5 kb hupL and 11 kb nifD elements during heterocyst differentiation required to activate the nitrogen fixation genes in Cyanobacteria.
*14 Chlorobium is a green sulfur bacterium: forma thiosulfatophilum; photoautotrophic growth on hydrogen sulfide and carbon dioxide.
CLEAR=ALL>
REFERENCES
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M. Mucke, V. Pingoud, G. Grelle, R. Kraft, D. H. Kruger, and M. Reuter Asymmetric Photocross-linking Pattern of Restriction Endonuclease EcoRII to the DNA Recognition Sequence J. Biol. Chem., April 12, 2002; 277(16): 14288 - 14293. [Abstract] [Full Text] [PDF]
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Y. Voziyanov, A. F. Stewart, and M. Jayaram A dual reporter screening system identifies the amino acid at position 82 in Flp site-specific recombinase as a determinant for target specificity Nucleic Acids Res., April 1, 2002; 30(7): 1656 - 1663. [Abstract] [Full Text] [PDF]
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S. E. Nunes-Duby, M. Radman-Livaja, R. G. Kuimelis, R. V. Pearline, L. W. McLaughlin, and A. Landy {lambda} Integrase Complementation at the Level of DNA Binding and Complex Formation J. Bacteriol., March 1, 2002; 184(5): 1385 - 1394. [Abstract] [Full Text] [PDF]
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K. P. Williams Integration sites for genetic elements in prokaryotic tRNA and tmRNA genes: sublocation preference of integrase subfamilies Nucleic Acids Res., February 15, 2002; 30(4): 866 - 875. [Abstract] [Full Text] [PDF]
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K. E. Shearwin, I. B. Dodd, and J. B. Egan The Helix-Turn-Helix Motif of the Coliphage 186 Immunity Repressor Binds to Two Distinct Recognition Sequences J. Biol. Chem., January 25, 2002; 277(5): 3186 - 3194. [Abstract] [Full Text] [PDF]
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A. Raynal, A. Friedmann, K. Tuphile, M. Guerineau, and J.-L. Pernodet Characterization of the attP site of the integrative element pSAM2 from Streptomyces ambofaciens Microbiology, January 1, 2002; 148(1): 61 - 67. [Abstract] [Full Text] [PDF]
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A. K. Sau, G. D. Tribble, I. Grainge, R. F. Frohlich, B. R. Knudsen, and M. Jayaram Biochemical and Kinetic Analysis of the RNase Active Sites of the Integrase/Tyrosine Family Site-specific DNA Recombinases J. Biol. Chem., November 30, 2001; 276(49): 46612 - 46623. [Abstract] [Full Text] [PDF]
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N. Messier and P. H. Roy Integron Integrases Possess a Unique Additional Domain Necessary for Activity J. Bacteriol., November 15, 2001; 183(22): 6699 - 6706. [Abstract] [Full Text] [PDF]
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T. J. D. Goodwin and R. T. M. Poulter The DIRS1 Group of Retrotransposons Mol. Biol. Evol., November 1, 2001; 18(11): 2067 - 2082. [Abstract] [Full Text] [PDF]
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D. Esposito, J. S. Thrower, and J. J. Scocca Protein and DNA requirements of the bacteriophage HP1 recombination system: a model for intasome formation Nucleic Acids Res., October 1, 2001; 29(19): 3955 - 3964. [Abstract] [Full Text] [PDF]
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 B. Thyagarajan, E. C. Olivares, R. P. Hollis, D. S. Ginsburg, and M. P. Calos Site-Specific Genomic Integration in Mammalian Cells Mediated by Phage {phi}C31 Integrase Mol. Cell. Biol., June 15, 2001; 21(12): 3926 - 3934. [Abstract] [Full Text]
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B. Casadewall, P. E. Reynolds, and P. Courvalin Regulation of Expression of the vanD Glycopeptide Resistance Gene Cluster from Enterococcus faecium BM4339 J. Bacteriol., June 1, 2001; 183(11): 3436 - 3446. [Abstract] [Full Text]
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S. A. Sciochetti, P. J. Piggot, and G. W. Blakely Identification and Characterization of the dif Site from Bacillus subtilis J. Bacteriol., February 1, 2001; 183(3): 1058 - 1068. [Abstract] [Full Text]
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E. Gindreau, R. López, and P. García MM1, a Temperate Bacteriophage of the Type 23F Spanish/USA Multiresistant Epidemic Clone of Streptococcus pneumoniae: Structural Analysis of the Site-Specific Integration System J. Virol., September 1, 2000; 74(17): 7803 - 7813. [Abstract] [Full Text]
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Q. Cheng, B. J. Paszkiet, N. B. Shoemaker, J. F. Gardner, and A. A. Salyers Integration and Excision of a Bacteroides Conjugative Transposon, CTnDOT J. Bacteriol., July 15, 2000; 182(14): 4035 - 4043. [Abstract] [Full Text]
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C. E. A. Peña, J. M. Kahlenberg, and G. F. Hatfull Assembly and activation of site-specific recombination complexes PNAS, June 23, 2000; (2000) 140014297. [Abstract] [Full Text]
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J. Wang, N. B. Shoemaker, G.-R. Wang, and A. A. Salyers Characterization of a Bacteroides Mobilizable Transposon, NBU2, Which Carries a Functional Lincomycin Resistance Gene J. Bacteriol., June 15, 2000; 182(12): 3559 - 3571. [Abstract] [Full Text]
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N. V. Grishin Two tricks in one bundle: helix-turn-helix gains enzymatic activity Nucleic Acids Res., June 1, 2000; 28(11): 2229 - 2233. [Abstract] [Full Text] [PDF]
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A. Gyohda and T. Komano Purification and Characterization of the R64 Shufflon-Specific Recombinase J. Bacteriol., May 15, 2000; 182(10): 2787 - 2792. [Abstract] [Full Text]
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L. S. Burns, S. G. J. Smith, and C. J. Dorman Interaction of the FimB Integrase with the fimS Invertible DNA Element in Escherichia coli In Vivo and In Vitro J. Bacteriol., May 15, 2000; 182(10): 2953 - 2959. [Abstract] [Full Text]
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G. J. Poelarends, L. A. Kulakov, M. J. Larkin, J. E. T. van Hylckama Vlieg, and D. B. Janssen Roles of Horizontal Gene Transfer and Gene Integration in Evolution of 1,3-Dichloropropene- and 1,2-Dibromoethane-Degradative Pathways J. Bacteriol., April 15, 2000; 182(8): 2191 - 2199. [Abstract] [Full Text]
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L. Jessop, T. Bankhead, D. Wong, and A. M. Segall The Amino Terminus of Bacteriophage lambda Integrase Is Involved in Protein-Protein Interactions during Recombination J. Bacteriol., February 15, 2000; 182(4): 1024 - 1034. [Abstract] [Full Text]
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F. Auvray, M. Coddeville, R. C. Ordonez, and P. Ritzenthaler Unusual Structure of the attB Site of the Site-Specific Recombination System of Lactobacillus delbrueckii Bacteriophage mv4 J. Bacteriol., December 1, 1999; 181(23): 7385 - 7389. [Abstract] [Full Text]
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T. Liu and E. Haggård-Ljungquist The Transcriptional Switch of Bacteriophage WPhi , a P2-Related but Heteroimmune Coliphage J. Virol., December 1, 1999; 73(12): 9816 - 9826. [Abstract] [Full Text]
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A. Petersen, J. Josephsen, and M. G. Johnsen TPW22, a Lactococcal Temperate Phage with a Site-Specific Integrase Closely Related to Streptococcus thermophilus Phage Integrases J. Bacteriol., November 15, 1999; 181(22): 7034 - 7042. [Abstract] [Full Text]
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S. Semsey, I. Papp, Z. Buzas, A. Patthy, L. Orosz, and P. P. Papp Identification of Site-Specific Recombination Genes int and xis of the Rhizobium Temperate Phage 16-3 J. Bacteriol., July 15, 1999; 181(14): 4185 - 4192. [Abstract] [Full Text]
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E. H. Cho, C.-E. Nam, R. Alcaraz Jr., and J. F. Gardner Site-Specific Recombination of Bacteriophage P22 Does Not Require Integration Host Factor J. Bacteriol., July 15, 1999; 181(14): 4245 - 4249. [Abstract] [Full Text]
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F. Guo, D. N. Gopaul, and G. D. Van Duyne Asymmetric DNA bending in the Cre-loxP site-specific recombination synapse PNAS, June 22, 1999; 96(13): 7143 - 7148. [Abstract] [Full Text] [PDF]
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S. Yang and L. K. Miller Activation of Baculovirus Very Late Promoters by Interaction with Very Late Factor 1 J. Virol., April 1, 1999; 73(4): 3404 - 3409. [Abstract] [Full Text]
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I. Grainge and D. J. Sherratt Xer Site-specific Recombination. DNA STRAND REJOINING BY RECOMBINASE XerC J. Biol. Chem., March 5, 1999; 274(10): 6763 - 6769. [Abstract] [Full Text] [PDF]
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C. E. A. Peña, J. M. Kahlenberg, and G. F. Hatfull Protein-DNA Complexes in Mycobacteriophage L5 Integrative Recombination J. Bacteriol., January 15, 1999; 181(2): 454 - 461. [Abstract] [Full Text]
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L. L. Voelker and K. Dybvig Characterization of the Lysogenic Bacteriophage MAV1 from Mycoplasma arthritidis J. Bacteriol., November 15, 1998; 180(22): 5928 - 5931. [Abstract] [Full Text]
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C.-J. Xu, Y.-T. Ahn, S. Pathania, and M. Jayaram Flp Ribonuclease Activities. MECHANISTIC SIMILARITIES AND CONTRASTS TO SITE-SPECIFIC DNA RECOMBINATION J. Biol. Chem., November 13, 1998; 273(46): 30591 - 30598. [Abstract] [Full Text] [PDF]
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A. Gravel, N. Messier, and P. H. Roy Point Mutations in the Integron Integrase IntI1 That Affect Recombination and/or Substrate Recognition J. Bacteriol., October 15, 1998; 180(20): 5437 - 5442. [Abstract] [Full Text]
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T. Bankhead and A. M. Segall Characterization of a Mutation of Bacteriophage lambda Integrase. PUTATIVE ROLE IN CORE BINDING AND STRAND EXCHANGE FOR A CONSERVED RESIDUE J. Biol. Chem., November 17, 2000; 275(47): 36949 - 36956. [Abstract] [Full Text] [PDF]
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A. C. Shaikh and P. D. Sadowski Trans Complementation of Variant Cre Proteins for Defects in Cleavage and Synapsis J. Biol. Chem., September 22, 2000; 275(39): 30186 - 30195. [Abstract] [Full Text] [PDF]
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B. O. Krogh and S. Shuman Vaccinia Topoisomerase Mutants Illuminate Conformational Changes during Closure of the Protein Clamp and Assembly of a Functional Active Site J. Biol. Chem., September 21, 2001; 276(39): 36091 - 36099. [Abstract] [Full Text] [PDF]
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L. Lee and P. D. Sadowski Directional Resolution of Synthetic Holliday Structures by the Cre Recombinase J. Biol. Chem., August 10, 2001; 276(33): 31092 - 31098. [Abstract] [Full Text] [PDF]
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G. Tribble, Y.-T. Ahn, J. Lee, T. Dandekar, and M. Jayaram DNA Recognition, Strand Selectivity, and Cleavage Mode during Integrase Family Site-specific Recombination J. Biol. Chem., July 14, 2000; 275(29): 22255 - 22267. [Abstract] [Full Text] [PDF]
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