DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination - PubMed (original) (raw)
DNA recognition, strand selectivity, and cleavage mode during integrase family site-specific recombination
G Tribble et al. J Biol Chem. 2000.
Abstract
We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.
Figures
FIG. 1. cis and trans cleavages within the Integrase family recombinases
A, the positioning of the catalytic Arg-His-Arg (RHR) triad and the tyrosine nucleophile around the scissile phosphodiester bond is shown. In the case of Flp (left), which cleaves DNA in trans, the RHR triad (shown in blue) is contributed by one protein monomer, and the tyrosine (shown in red) is contributed by a second monomer. For Cre (right), the triad as well as the tyrosine are derived from a single Cre monomer (all residues shown in blue). The arrangement of the phosphate group and the amino acid side chains are redrawn from the Cre-DNA crystal structure (10). B, a recombinase dimer that cleaves in cis (I; Cre, for example) can, in principle, be related to one that cleaves in trans (Flp, for example) in one of two ways (II or III). The tyrosine (from the green monomer) that cleaves at the left end of the spacer can be pushed to the right end in its functional orientation as shown in II. Or, the tyrosine from the monomer at the right (purple) can be moved to the left end as shown in III. The cleavage susceptible phosphodiester positions are indicated by the dots. The representation of the cleavage active and inactive recombinase monomers in green and purple, respectively, in the cis cleaving dimer (I) follows the color codes used by Guo et al. (10).
FIG. 2. Synthetic DNA substrates and tagged variants of Flp used in the Flp footprinting assays
A, the sequences of the individual substrates are shown with the Flp-binding elements written in bold uppercase letters, and the arrows indicate their relative orientations. The linear substrates S1 and S2 and the Holliday junction HS4 contain one, two, and four Flp-binding elements, respectively. The Holliday junction is represented in an antiparallel configuration. Note that the two Flp-binding elements within a recombination substrate (say, S2) differ in one of the 13-bp positions; the nearest neighbors of the GC pair immediately flank the strand exchange region. It is a TA pair in the left binding element of S2 and an AT pair in the right binding element. Interaction between two Flp monomers, one bound at the left and one bound at the right of the 8-bp spacer, is essential for the assembly of a functional strand cleavage pocket. The non-binding substrate NS was obtained by altering 12 positions each within the left and right binding elements. The defunct binding elements are shown in italics. B, the wild type Flp protein was tagged at either the amino terminus with the T7 tag (row 1) or at both the amino and carboxyl termini with the T7 and S tag (row 2), respectively. Flp(Y343F) was tagged at the amino terminus with a combined His6-S tag (row 3). The sequences of the tags and the additional mass contributed by them to the 46.5 kDa of native Flp are indicated.
FIG. 3. Chymotryptic footprint patterns of N-tagged Flp or Flp(Y343F) and C-tagged Flp
The reactions in A–D were carried out in identical manner (“Materials and Methods”). The samples in A and B were electrophoretically fractionated for different lengths of time to highlight individual features within the footprints. The Western blots were probed with a monoclonal antibody against the T7 peptide in A and B and with the S probe in C and D. The N-C ruler placed at the right of each panel measures the distance of a protected region from the tagged end. Lanes 1 and 2 are untreated Flp and Flp treated with chymotrypsin in the absence of DNA. S1, S2, HS4, and NS refer to the presence of substrates with one, two, four, and no Flp-binding elements (see Fig. 2_A_ for details), respectively, in the protease reactions. Regions of protection for the N-tagged proteins are marked P with an appropriate suffix. The P’ patches refer to the footprints from the C-tagged protein. The suffixes I–IV match them to the P footprints from the N-tagged protein. The arrows with the p’ label (p’0 and p’5 in C) indicate protected bands whose counterparts from the N-tagged profile were not apparent. The band migrating above the Flp band (CL in A and B) is due to the covalent attachment of Flp to DNA as a result of strand cleavage. E refers to enhanced chymotryptic cleavage in _lanes 3–5. A_’ represents a longer exposure of the luminogram of A to accentuate features of the PII footprint.
FIG. 4. Footprinting profiles of N-tagged Flp with trypsin and proteinase K, distribution of protected regions on the Flp protein
A and B, the footprints of N-tagged Flp using trypsin and proteinase K are shown. Lane designations and other details are as in Fig. 3. The probe was a monoclonal antibody directed against the T7 peptide. The PII footprint observed with chymotrypsin (see Fig. 3) was also revealed by trypsin (A). Two protected bands p3 and p5 were displayed by proteinase K (B). The Flp-DNA covalent adduct is labeled CL. C, the Flp protein is schematically represented as a cylinder. The cleavage protection (denoted by _P, p_’) and enhancement (denoted by E) data from Fig. 3 are mapped on Flp after compensating for the sizes of the tags used for end labeling (see Fig. 2; also explanation in text). The approximate ranges of PI, PII, and PIII, based on molecular mass estimates, are 114–150, 186–259, and 295–314, respectively. Potential chymotryptic sensitive sites near the PI borders are Tyr-107, Tyr-108, and Trp-155. The amino-terminal PII border matches Phe-182, Phe-186, or Phe-192. Phe-248 or Tyr-271 form the likely carboxyl-terminal border of PII. Tyr-293, Phe-296, and Phe-314 are plausible residues that delimit PIII. The protease-sensitive sites in Flp or Flp(Y343F) adjacent to or overlapping a subset of the protected regions were determined by amino-terminal microsequencing (this study; see Ref. 8).
FIG. 5. Selective strand cleavage by Cre and Flp in substrates containing strand-specific bulges
A, the Cre and Flp substrates (at left and right, respectively) are schematically shown with their spacer sequences written out, and the scissile phosphodiesters indicated as p. The recombinase binding elements are indicated by the parallel arrows that also represent their head to head orientation. The top and bottom strands (arbitrary designations) are distinguished by thick and thin lines, respectively. The spacer positions are numbered from left to right on the top strand and from right to left on the bottom strand (in the 5′ to 3′ direction). The A3 bulges were introduced between positions 3 and 4 or 3′ and 4′ in the Cre substrate and between 4 and 5 or 4′ and 5′ in the Flp substrate. The reactions were carried out on substrates labeled with 32P at the 3′ ends on both strands (indicated by the asterisks). The bands CL and CR represent cleavage fragments derived from the left and right ends of the spacer, respectively. The cleavage bands from the bulge containing strands would be 3 nucleotides longer than those from the control substrate. The unreacted substrate bands are denoted by S. The presence of more than one cleavage band at a given spacer end is due partly to substrate heterogeneity and partly to the occurrence of an occasional aberrant cleavage in linear substrates. B, the experimental results from A are schematically diagrammed at the bottom. The Cre and Flp substrates are drawn in thick and thin lines, respectively; the corresponding recombinase proteins are represented by the unshaded and shaded ovals, respectively. The phosphodiester that is reactive during a cleavage event is shown by the filled circle. The unreactive one is shown by the open circle.
FIG. 6. Strand cleavage by Flp(H305L) and Flp in substrates with spacer bulges and spacer mismatches
A, in the normal Flp substrate shown at the top, the numbers 0 and 0′ (C) and 9 and 9′ (G) refer to the nucleotide positions immediately flanking the spacer. The bulge positions on the top strand are denoted above the respective reactions. The reactions in lanes 2 and 3 were done with the control substrate harboring no spacer bulges. B, in the schematic representation of Flp substrates, the mismatched nucleotides are indicated. The designations of substrate and cleavage bands in A and B follow the symbols used in Fig. 5.
FIG. 7. The cleavage yields by Flp(H305L) from Fig. 6_A_, CL and CR, respectively, were calculated (as described under “Materials and Methods”) and graphed as a function of the bulge position (indicated by the vertical arrows on the abscissa)
The cleavage curve was drawn (solid line) by connecting points representing the algebraic sum of CL and CR for each bulge position. The values of CL for the bulge placed between 0 and 1 and that of CR for the bulge placed between 8 and 9 were obtained from the Flp reactions (lanes 5 and 14 of Fig. 6_A_). For a detailed explanation, see text. This manipulation was necessitated by the severely diminished cleavage capacity of Flp(H305L) at a scissile phosphodiester position when it was flanked immediately by a bulge on the same strand or the opposite strand (lanes 6 and 15 of Fig. 6_A_; see also text for details). The dashed line is the cleavage curve computed for bottom strand bulges from an experiment analogous to that shown in Fig. 6_A_.
FIG. 8. Strand cleavage bias as a function of the bulge position within the Cre spacer
A, the bulge locations are indicated above the respective reactions. A control assay with the normal substrate (schematically depicted at the top) is shown in lane 2. B, the amounts of CL and CR products from reactions with the bulge-containing substrates were normalized against the corresponding amounts from the control substrate (lane 2) as described under “Materials and Methods.” The graph represents the averaged values from two reaction sets, one of which is shown in A.
FIG. 9. Comparison of Flp footprints to DNA contacts observed in the crystal structure of Cre bound to DNA; a model for DNA recognition by Flp
A, the secondary structure representation of the carboxyl-terminal domain of Cre is taken from Guo et al. (10) and that of Flp is based on the Protein Secondary Structure Prediction Program provided by the Baylor College of Medicine, Houston. The predicted Flp helices (bottom panel) are named arbitrarily to obtain reasonable matches with the Cre nomenclature. The asterisks in Cre indicate amino acids that are in close proximity to DNA. The shaded sequences in Cre represent two conserved regions in prokaryotic Int type recombinases as follows: Box I (or Box A) and Box II (or Box B plus Box C) (1, 2). In Flp, the regions of protease protection are underlined and labeled as in Fig. 4_C_. The conserved boxes of the yeast family recombinases, Box I and Box II (41), are shown by the shaded areas. Note that the demarcation of the yeast family Box II places the active site tyrosine (shown in bold letter) outside its boundary, whereas the prokaryotic Box II includes this residue. The RHR catalytic triads (within Box I and Box II) are also shown in bold letters. The observed protections in Flp are in fair agreement with the expectations based on the protein-DNA and protein-protein contacts revealed by the Cre structure (10). The Flp to Cre alignment is similar to those published previously (2, 12). B, the global three-dimensional topology of the Flp protein is obtained by fitting the secondary structure of Flp (predicted by Protein Secondary Structure Prediction Program) to a proposed model structure for Flp (30). To do this, the amino-terminal domain of the protein is moved with respect to the carboxyl-terminal domain using the presumably flexible interdomainal peptide loop so as to bring the former into close proximity with DNA. In the resulting protein-DNA model, the DNA is held between the two domains in a manner that is analogous to the mode of substrate association by the Cre protein (10). The peptide regions protected in the protease footprinting assays (p_’_0, PI–IV) are indicated in red. Note that the A/B helices together with the D helix could form a scissors-like grip on the DNA primarily through the major groove. The clustering of the RHR triad residues around the scissile phosphodiester bond (shown in red) and the positioning of the active site tyrosine (shown in green) away from it are consistent with the mechanism of phosphate orientation and the trans mode of nucleophile donation during strand cleavage by Flp. The protection of this tyrosine and the adjacent peptide segment must result from proximity to DNA at the other end of the spacer.
FIG. 10. Peptide connectivities that determine cis or trans cleavage by a recombinase dimer
Top, perspective of the cleaved Cre-DNA complex (10) as viewed from a vantage point on the carboxyl-terminal side of the protein is shown. The DNA bend corresponds to a bottom strand bulge near the center of the spacer. Cleavage has occurred at the right-hand side, executed in the cis mode by the Cre monomer in green. Bottom, the same perspective as in the top panel is redrawn at the left to represent the helices K–N and their linkages. At the right, the change in connectivity between the purple and green monomers to mediate trans cleavage is shown. Effectively, the purple L helix is linked to the green M’–N’ helices. Conversely, the green L’ helix is linked to the purple M–N helices. The new location of the M–N helices approximates the situation for the recombinase tetramer seen in DNA-protein cocrystal structures (23).
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