Phosphate coordination and movement of DNA in the Tn5 synaptic complex: role of the (R)YREK motif

Abstract

Bacterial DNA transposition is an important model system for studying DNA recombination events such as HIV-1 DNA integration and RAG-1-mediated V(D)J recombination. This communication focuses on the role of protein-phosphate contacts in manipulating DNA structure as a requirement for transposition catalysis. In particular, the participation of the nontransferred strand (NTS) 5' phosphate in Tn5 transposition strand transfer is analyzed. The 5' phosphate plays no direct catalytic role, nonetheless its presence stimulates strand transfer approximately 30-fold. X-ray crystallography indicates that transposase-DNA complexes formed with NTS 5' phosphorylated DNA have two properties that contrast with structures formed with complexes lacking the 5' phosphate or complexes generated from in-crystal hairpin cleavage. Transposase residues R210, Y319 and R322 of the (R)YREK motif coordinate the 5' phosphate rather than the subterminal NTS phosphate, and the 5' NTS end is moved away from the 3' transferred strand end. Mutation R210A impairs the 5' phosphate stimulation. It is posited that DNA phosphate coordination by R210, Y319 and R322 results in movement of the 5' NTS DNA away from the 3'-end thus allowing efficient target DNA binding. It is likely that this role for the newly identified RYR triad is utilized by other transposase-related proteins.

Figure 1.

Steps of Tn5 transposition. A scheme depicting distinct Tn5 transposition steps and corresponding DNA-protein species that are as follows. (Step 1) ‘The initial substrate’. Transposon end sequence (ES) DNA is found adjacent to donor backbone (dbb) DNA; (Step 2) ‘The nicked substrate’. Transposon ES DNA-donor DNA containing a precise nick exposing a 3′ hydroxyl at the end of the ES DNA transferred strand (TS); (Step 3) ‘Hairpin formation’. Transposon ES DNA in which a hairpin links the transferred strand (TS) and non-transferred strand (NTS); (Step 4) ‘Hairpin resolution’. Transposon ES DNA with a blunt cleaved end resulting from resolution of the hairpin; (Step 5) ‘Target capture’. A complex that contains both cleaved ES DNA and a target DNA sequence; (Step 6) ‘Strand transfer’. A complex in which the ES TS has been covalently linked to one strand of the target DNA to generate the final product. Also shown are nucleophilic species involved in sequential catalytic steps.

Figure 2.

The nontransferred strand 5′ phosphate stimulates strand transfer. (A) Paired end complexes containing fluorescently labeled end sequence DNA, whose NTS contained either a 5′ hydroxyl or a 5′ phosphate, were mixed with super-coiled DNA in the presence of Mg²⁺. The combined steps of target capture and strand transfer were visualized by agarose gel analysis of reaction products from different time points after the start of the reaction. The bottom band (PEC) represents unreacted paired end complexes. The SEST band represents strand transfer of a single ES into the plasmid DNA forming open circle molecules. The double end strand transfer (DEST) band represents the final DNA transposition event in which both end sequences are transferred into the plasmid DNA forming linear molecules. The NTS 5′ phosphate stimulates both SEST and DEST product formation. (B) Summary of the quantitative analysis of gels similar to the one shown in Figure 2A. The time course of DEST product formation for the 5′ hydroxyl (open circles) and 5′ phosphate end sequence DNA substrates (closed circles) is compared using data derived from the gels similar to that presented in Figure 2A. Error bars represent standard errors of the triplicate experimental points. Solid lines represent linear regressions calculated using the data in the linear range of the kinetic response. The difference between these calculated rates of the DEST formation is 32-fold. Qualitatively similar results are obtained by summing the SEST and DEST data band intensities.

Figure 3.

Effect of 5′ phosphate on DNA structure in the Tnp paired end complexes. The DNA structure for the paired end complex containing phosphorylated NTS reported here (colored by atom type in blue, cyan, red and orange) is compared to the DNA structure reported previously for the paired end complex lacking 5′ phosphate (PDB code 1MUH, in yellow) in a stereo presentation. The arrow shows the change of the terminal oxygen position between the two structures. The result of this movement is a near perfect overlap between the 5′-terminal phosphate and a phosphate bridging bases 1 and 2 in the nonphysiological NTS 5′ OH structure. Note the corresponding movement of the base 1 of the NTS and a flipped over base 1 of the TS. The combined effect is to provide more room in the vicinity of the catalytically active 3′ OH of the TS and that this increased room facilitates the binding of target DNA in preparation for strand transfer.

Figure 4.

5′ Phosphate coordination by Tnp. Coordination of the NTS 5′ phosphate by Tnp amino acid residues R210, Y319 and R322 is illustrated. NTS is shown in green and TS in cyan. In both cases noncarbon atoms of base 1 of both strands are colored according to atom type. For clarity, DDE catalytic core is not shown. Catalytic E326 is located one helical turn away from R322.

Figure 5.

Strand transfer by Tnp R210A Mutant. The Tnp R210A catalyzed strand transfer activity was determined exactly as outlined in Figure 2. Shown are representative gels containing SEST and double end strand transfer (DEST) reaction products and a graph that presents quantitative analysis of the kinetic data derived from the gels. The time course over a period of 6 h is plotted for the mutant Tnp in complex with 5′ hydroxyl (open squares) or 5′ phosphate (closed squares) end sequence DNA substrates. Data for the control Tnp in complex with 5′ hydroxyl end sequence DNA substrate (open circles) is plotted for comparison. Error bars represent standard errors of the triplicate data points for R210A mutant and of the duplicates for the control. Solid lines represent linear regressions through all data presented. For Tnp R210A, the difference in the calculated reaction rates between NTS 5′ P and 5′ OH is 1.8-fold. Thus, Tnp R210A displays impaired strand transfer activity in comparison to the control, but the stimulatory effect of the 5′ phosphate on strand transfer activity with this mutant is only ∼2-fold.

Figure 6.

Effect of 5′ phosphate on accessibility of the catalytic site in the Tn5 paired end complex. The structure of the paired end complex containing phosphorylated NTS (right) is compared to the previously reported structure lacking the 5′ phosphate (PDB code 1MUH). Shown are molecular surface representations with protein colored gray and DNA colored yellow. The first base of the nontransferred DNA strand is colored green and the 3′ catalytic hydroxyl of the transferred strand is colored red. Note how in the presence of the 5′ phosphate the movement of the DNA illustrated in Figure 3 creates a cavity in the active site exposing the 3′ OH. Presumably the DNA movement eliminates steric hindrances for the target DNA binding in the subsequent step of DNA transposition.

Figure 7.

A new step in Tn5 transposition—rearrangement of cleaved DNA prior to target capture. A portion of the schematic presented in Figure 1 is modified to accommodate the results from the current studies. Step (4) has been modified to show the R210, Y319, R322 (RYR) coordination to the phosphate bridging bases 1 and 2 of the NTS immediately following hairpin cleavage. (4a) The NTS 5′-end is then rearranged through the formation of RYR coordination to the 5′-terminal phosphate and the NTS 5′-end is displaced from the 3′-end thus facilitating formation of the target capture complex [step (5)].