Binding and catalytic contributions to site recognition by flp recombinase

Abstract

Flp catalyzes site-specific recombination in a highly sequence-specific manner despite making few direct contacts to the bases within its binding site. Sequence discrimination could take place in the binding and/or the catalytic steps. In this study, we independently measure the binding affinity and initial cleavage rate of Flp recombinase with approximately 20 designed alternate target DNA sequences. Our results show that Flp specificity is largely, although not entirely, imparted at the binding step and is the result of a combination of direct and indirect readout. The Flp binding site includes an A/T-rich region that displays a characteristically narrow minor groove. We find that many A --> T changes are tolerated at the binding step, whereas C or G substitutions tend to decrease binding affinity. The effects of the latter can be alleviated by replacing guanine with inosine, which removes the N2 amino group that protrudes into the minor groove. Some A --> T changes reduce binding affinity, due to clashing with nearby residues, reinforcing that specificity requires avoiding negative contacts as well as creating positive ones. A tracts, which can lead to unusually rigid DNA structure, are tolerated during the binding step when placed within the region where the minor groove is already narrow. However, most A tracts slow catalysis more than C or G substitutions. Understanding what kind of sequence variation is tolerated in the binding and catalytic steps helps us understand how the target DNA is recognized by Flp and will be useful in guiding the design of Flp variants with altered specificities.

FIGURE 1.

Flp–DNA interactions. A, a Holliday junction-bound Flp tetramer. The active monomers are yellow, the inactive monomers are blue, and the catalytic tyrosines (Tyr-343) are red. B, the DNA from a Flp tetramer, with the scissile phosphates marked by orange spheres. C, closeup view of Flp interacting with the arm highlighted in B. Green side chains are in proximity to DNA in D. A different view of the same interactions shown in C, with an A to T transversion modeled in position 3 (pink). The methyl group of T clashes with Ala-55 (green). Also shown is Met-58, which is poorly ordered but lies near the hydrophobic methyl group of T5. A was adapted from Ref. , using Protein Data Bank code 1FLO (28), and parts B–D made with PyMol (29), PDB 1M6X (22).

FIGURE 2.

The FRT sequence. A, the full FRT site, with arrows marking the 3 Flp-binding subsites. Positions discussed in the text are numbered. B, the WT FRT suicide substrate used in binding and cleavage assays (variations are shown in Table 1). Vertical arrow marks the cleavage site, and several Flp side chains are shown next to the bases they are in close proximity to (see Fig. 1).

FIGURE 3.

Analysis of the Flp-bound DNA structure. A, minor groove width. Note that it becomes narrow in the A/T-rich region near the scissile phosphate. The dashed line represents the average minor groove width for canonical B DNA. B, roll angles between base pairs. Both plots were made using 3DNA version 1.5 (30). Three examples of the Flp binding site were taken from structures determined in two different space groups (PDB codes 1FLO and 1M6X).

FIGURE 4.

Competition binding assays. A, example of a competition assay native gel, and B–E, plots of competition assays with FRT variants containing scissile phosphate position changes (B), A tracts (C), A to T transversions (D), and C or G substitutions (E). The fraction of radiolabeled WT FRT that is bound to Flpe Y343F decreases with increasing amounts of unlabeled competitor FRT DNA. Lines represent fitting the average and standard deviation of 3–6 experiments to an equation for the IC₅₀ (shown under “Experimental Procedures”).

FIGURE 5.

Summary of catalysis data. Rate constants are plotted in order of binding affinity, as indicated by the arrow and K_i values at the left. The sequences of the FRT substrates on the y axis are shown in Table 1.

FIGURE 6.

Diagram showing A:T and G:C base pairs, along with the modified bases of uridine and inosine shown in A:U and I:C base pairs. Uridine lacks the methyl group that may cause a steric clash with Ala-55 in the Frt3at substrate; a uridine-containing substrate (Frt3au) had much higher affinity for Flp. Inosine lacks the amine group of guanosine that normally prevents propeller twisting and narrowing of the minor groove. Note that the minor groove (lower) face of an I:C base pair also mimics that of an A:T base pair.

FIGURE 7.

Comparison of Flp-bound DNA structures before and after cleavage. A, uncut, and B, after cleavage. The scissile phosphate kinks in, forming an unusual structure. The ability to form this kinked conformation may be necessary for catalysis.