An Enzyme-Catalyzed Multistep DNA Refolding Mechanism in Hairpin Telomere Formation

<div><p>Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting–rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.</p> </div

The enzyme active site.

<p>Orthovanadate bridges the DNA 5′-OH group, 3′-OH group, and the Tyr405 sidechain in the wild-type TelA hairpin–vanadate complex. The magenta sphere in the middle represents vanadium in the active site. The active site of TelA is similar to those of type-IB topoisomerases and tyrosine recombinases. But TelA is unique in having Tyr at residue 363 (commonly Lys in topoisomerases and His in tyrosine recombinases). The <i>Borrelia</i> protelomerase/telomere resolvase ResT also has Tyr at this position <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001472#pbio.1001472-Deneke2" target="_blank">[32]</a>. Simulated annealing composite omit 2Fo-Fc densities within 2.0 Å from the DNA or active site protein atoms are shown, contoured at 1.5 σ (blue) or 7.0 σ (red).</p

The strand-refolding intermediate.

<p>(A) The open DNA conformation observed in the phosphotyrosine complexes stabilized by stacking of the flipped-out bases, G-T wobble base-pairs, and water-mediated hydrogen bonds. The protein and DNA molecules are colored as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001472#pbio-1001472-g004" target="_blank">Figure 4A</a>. (B) Schematic diagram of the strand-refolding intermediate DNA conformation. The 5′ bases Thy1, Cyt2, and Ade3 are flexible. Thy1 in our crystal structures is either missing or replaced by cytosine to block ligation and trap the phosphotyrosine bond. (C) Simulated annealing omit Fo-Fc electron density contoured at 3.0 σ for the G-T wobble pair. (D) 5′-overhang conformation observed in the phosphotyrosine complexes (the refolding intermediate) trapped using two different types of suicide DNA substrates. The structure obtained with the nicked suicide substrate (DNAb/DNAc) is shown in yellow, while that obtained with a mismatch-based suicide substrate (DNAd) is shown in purple. The tri-nucleotide stretch Thy4, Gua5, and Ade6 is superimposable with an r.m.s.d. of 0.78 Å. The deviation comes mostly from the phosphate backbone atoms. (E) Comparison of the DNA structures between the refolding intermediate (yellow) and the hairpin telomere product (alternate conformations shown in black and grey). The superposition is based on the double-stranded stem region of the DNAs.</p

Structure of the hairpin telomere bound to TelA.

<p>(A) The compact di-nucleotide hairpin DNA product. All bases except two at the apex (Ade3 and Thy4) form Watson–Crick base-pairs. The DNA strand near the tip of the hairpin loop adopts two alternative conformations as indicated by the red arrows. The two protein subunits and two DNA molecules in the TelA–DNA complex are all colored differently (yellow/green for DNA, and grey/cyan for protein) to highlight <i>cis</i> versus <i>trans</i> interactions made by TelA. (B) Schematic diagram of the hairpin DNA conformation. The red dots represent the scissile phosphates. (C and D) Simulated annealing composite omit 2Fo-Fc densities for the wild-type TelA–hairpin DNA complex (C) and the R255A unligated hairpin complex (D). Electron densities within 2.0 Å from the DNA or active site protein atoms are shown, contoured at 1.5 σ (blue) or 7.0 σ (red) above the mean levels. (E) Two hairpin ends in alternative conformations are packed tightly across the 2-fold axis, interacting with one another as well as with Arg205. Van der Waals radii for Arg205 and Thy4 are shown by dots.</p

Schematic diagram and close-up views of the TelA–hairpin DNA interactions.

<p>Solid lines and dashed lines denote direct and indirect (water-mediated) interactions, respectively. The close-up views in the insets show interactions involving the phosphate backbones in the hairpin loop. The 2Fo-Fc electron densities are contoured at 1.5 σ.</p

Requirements for the formation of hairpin products.

<p>(A) In vitro hairpin formation assay on the wild-type and mutant TelA proteins, showing that the non-active site residues Tyr201 and Arg205 are essential in hairpin product formation. Tyr405 is the catalytic nucleophile residue that forms the phosphotyrosine bond. (B) 5′-overhang conformations for mutant TelA–DNA complexes. The tri-nucleotide stretch including Thy4, Gua5, and Ade6 is shown for the refolding intermediate (phosphotyrosine complex formed with the wild-type TelA) in yellow, the DNA complexed with R205A in blue, and that complexed with Y201A in red. (C) In vitro hairpin formation by the wild-type TelA on DNA substrates with various 6 bp palindromic sequences between the scissile sites. Duplicated samples are with separate substrate DNA preparations.</p

Overall structure of the TelA dimer bound to hairpin telomeres.

<p>Molecular surface is shown for one of the molecules in the TelA dimer, whereas the other molecule is shown by ribbons. The view in (A) is perpendicular to and that in (B) is parallel to the 2-fold axis relating the two TelA molecules. The two hairpin DNAs in alternative conformations are shown, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001472#pbio-1001472-g004" target="_blank">Figure 4E</a>.</p

X-ray data collection, phasing, and refinement statistics.

<p>Statistics for the highest resolution shell are shown in parentheses.</p

Sequence-specific contacts near the hairpin end.

<p>Sequence-specific interactions by Lys208 and Lys288 that stabilize the 5′-terminal Thy1 base in the hairpin product bound to TelA. The magenta sphere in the very bottom of the image represents vanadium in the active site. Two alternative conformations are shown for the Lys208 sidechain.</p

Hairpin telomere generation by protelomerase TelA.

<p>(A) Schematic diagram showing how a bacterial linear chromosome with hairpin telomeres is replicated <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001472#pbio.1001472-Picardeau1" target="_blank">[6]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001472#pbio.1001472-Ravin1" target="_blank">[7]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001472#pbio.1001472-Chaconas1" target="_blank">[10]</a>. The last step is resolution of concatenated chromosomes at the inverted repeat junctions, which is carried out by protelomerase (also known as telomere resolvase). (B) A model for the hairpin telomere formation by the protelomerase TelA, based on the crystal structures presented in this study. A key feature of the reaction mechanism of TelA revealed by this study is the “refolding intermediate” conformation that is stabilized by a set of transient protein–DNA and DNA–DNA interactions.</p