Anne-Cécile Déclais

Structural dynamics of individual Holliday junctions

The four-way DNA (Holliday) junction is the central intermediate of genetic recombination, but the dynamic aspects of this important structure are presently unclear. Although transitions between alternative stacking conformers have been predicted, conventional kinetic studies are precluded by the inability to synchronize the junction in a single conformer in bulk solution. Using single-molecule fluorescence methodology we have been able to detect these transitions. The sequence dependence, the influence of counterions and measured energetic barriers indicate that the conformer transition and branch migration processes share the unstacked, open structure as the common intermediate but have different rate-limiting steps. Relative rates indicate that multiple conformer transitions occur at each intermediate step of branch migration, allowing the junction to reach conformational equilibrium. This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution.

Cooperative Control of Holliday Junction Resolution and DNA Repair by the SLX1 and MUS81-EME1 Nucleases

Summary Holliday junctions (HJs) are X-shaped DNA structures that arise during homologous recombination, which must be removed to enable chromosome segregation. The SLX1 and MUS81-EME1 nucleases can both process HJs in vitro, and they bind in close proximity on the SLX4 scaffold, hinting at possible cooperation. However, the cellular roles of mammalian SLX1 are not yet known. Here, we use mouse genetics and structure function analysis to investigate SLX1 function. Disrupting the murine Slx1 and Slx4 genes revealed that they are essential for HJ resolution in mitotic cells. Moreover, SLX1 and MUS81-EME1 act together to resolve HJs in a manner that requires tethering to SLX4. We also show that SLX1, like MUS81-EME1, is required for repair of DNA interstrand crosslinks, but this role appears to be independent of HJ cleavage, at least in mouse cells. These findings shed light on HJ resolution in mammals and on maintenance of genome stability.

TRF2 promotes, remodels and protects telomeric Holliday junctions

The ability of the telomeric DNA‐binding protein, TRF2, to stimulate t‐loop formation while preventing t‐loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t‐loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t‐loops and at regressed replication forks.

Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I

We have solved the crystal structure of the Holliday junction resolving enzyme T7 endonuclease I at 2.1 Å resolution using the multiwavelength anomalous dispersion (MAD) technique. Endonuclease I exhibits strong structural specificity for four-way DNA junctions. The structure shows that it forms a symmetric homodimer arranged in two well-separated domains. Each domain, however, is composed of elements from both subunits, and amino acid side chains from both protomers contribute to the active site. While no significant structural similarity could be detected with any other junction resolving enzyme, the active site is similar to that found in several restriction endonucleases. T7 endonuclease I therefore represents the first crystal structure of a junction resolving enzyme that is a member of the nuclease superfamily of enzymes.

The Caenorhabditis elegans Homolog of Gen1/Yen1 Resolvases Links DNA Damage Signaling to DNA Double-Strand Break Repair

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53–mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.

The structural basis of Holliday junction resolution by T7 endonuclease I.

The four-way (Holliday) DNA junction is the central intermediate in homologous recombination, a ubiquitous process that is important in DNA repair and generation of genetic diversity. The penultimate stage of recombination requires resolution of the DNA junction into nicked-duplex species by the action of a junction-resolving enzyme, examples of which have been identified in a wide variety of organisms. These enzymes are nucleases that are highly selective for the structure of branched DNA. The mechanism of this selectivity has, however, been unclear in the absence of structural data. Here we present the crystal structure of the junction-resolving enzyme phage T7 endonuclease I in complex with a synthetic four-way DNA junction. Although the enzyme is structure-selective, significant induced fit occurs in the interaction, with changes in the structure of both the protein and the junction. The dimeric enzyme presents two binding channels that contact the backbones of the junction’s helical arms over seven nucleotides. These interactions effectively measure the relative orientations and positions of the arms of the junction, thereby ensuring that binding is selective for branched DNA that can achieve this geometry.

Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I.

T7 endonuclease I is a nuclease that is selective for the structure of the four‐way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild‐type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion‐binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 ± 0.019 and 14 ± 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1‐bound metal ion.

The complex between a four-way DNA junction and T7 endonuclease I.

The junction‐resolving enzyme endonuclease I is selective for the structure of the DNA four‐way (Holliday) junction. The enzyme binds to a four‐way junction in two possible orientations, with a 4:1 ratio, opening the DNA structure at the centre and changing the global structure into a 90° cross of approximately coaxial helices. The nuclease cleaves the continuous strands of the junction in each orientation. Binding leads to pronounced regions of protection of the DNA against hydroxyl radical attack. Using all this information together with the known structure of the enzyme and the structure of the BglI–DNA complex, we have constructed a model of the complex of endonuclease I and a DNA junction. This shows how the enzyme is selective for the structure of a four‐way junction, such that both continuous strands can be accommodated into the two active sites so that a productive resolution event is possible.

Single-molecule studies of DNA and RNA four-way junctions

Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.

Crystal Structure of a Eukaryotic GEN1 Resolving Enzyme Bound to DNA

Summary We present the crystal structure of the junction-resolving enzyme GEN1 bound to DNA at 2.5 Å resolution. The structure of the GEN1 protein reveals it to have an elaborated FEN-XPG family fold that is modified for its role in four-way junction resolution. The functional unit in the crystal is a monomer of active GEN1 bound to the product of resolution cleavage, with an extensive DNA binding interface for both helical arms. Within the crystal lattice, a GEN1 dimer interface juxtaposes two products, whereby they can be reconnected into a four-way junction, the structure of which agrees with that determined in solution. The reconnection requires some opening of the DNA structure at the center, in agreement with permanganate probing and 2-aminopurine fluorescence. The structure shows that a relaxation of the DNA structure accompanies cleavage, suggesting how second-strand cleavage is accelerated to ensure productive resolution of the junction.