Biomedical subjects

Anne-Cécile Déclais

Publications and source records attributed to Anne-Cécile Déclais.

7 recordsLinked to original sources

PubMed2006

Structural recognition between a four-way DNA junction and a resolving enzyme.

Resolving enzymes bind highly selectively to four-way DNA junctions, but the mechanism of this structural specificity is poorly understood. In this study, we have explored the role of interactions between the dimeric enzyme and the helical arms of the junction, using junctions with either shortened arms, or circular permutation of arms. We find that DNA-protein contacts in the arms containing the 5' ends of the continuous strands are very important, conferring a significant level of sequence discrimination upon both the choice of conformer and the order of strand cleavage. We have exploited these properties to obtain hydroxyl radical footprinting data on endonuclease I-junction complexes that are not complicated by the presence of alternative conformers, with results that are in good agreement with the arm permutation and shortening experiments. Substitution of phosphate groups at the center of the junction reveals the importance of electrostatic interactions at the point of strand exchange in the complex. Our data show that the form of the complex between endonuclease I and a DNA junction depends on the core of the junction and on interactions with the first six base-pairs of the arms containing the 5' ends of the continuous strands.

Bacteriophage T7↗

PubMed2006

Mechanistic aspects of the DNA junction-resolving enzyme T7 endonuclease I.

The chemical mechanism of phosphodiester bond hydrolysis catalyzed by a junction-resolving enzyme has been investigated. Endonuclease I of phage T7 is a member of the nuclease superfamily of proteins that include many restriction enzymes, and the structure of the active site is very similar to that of BglI in particular. It contains three acidic amino acids that coordinate two divalent metal ions. Using mass spectrometry we have shown that endonuclease I catalyzes the breakage of the P-O3' bond, in common with restriction enzymes. We have found that the pH dependence of the hydrolysis reaction is log-linear, with a gradient of 0.9. Substitution of the scissile phosphate by an electrically neutral methylphosphonate significantly impairs the rate of bond cleavage. However, the introduction of chirally pure methylphosphonate groups shows that the effect of substitution of the proS oxygen atom is much greater than that for the proR. This is consistent with our current model of the structure of the DNA bound in the active site of endonuclease I, where the proS oxygen atom is coordinated directly to both metal ions as it is in BglI. The activity is also very sensitive to repositioning of the carboxylate groups of Asp 55 and Glu 65 in the active site, although some restoration of activity in endonuclease I E65D was observed in the presence of Mn2+ ions. A mechanism of hydrolysis consistent with all of these data is proposed.

Base Sequence↗

PubMed2005

Stereospecific effects determine the structure of a four-way DNA junction.

Conversion of a centrally located phosphate group to an electrically neutral methyl phosphonate in a four-way DNA junction can exert a major influence on its conformation. However, the effect is strongly dependent on stereochemistry. Substitution of the proR oxygen atom by methyl leads to conformational transition to the stacking conformer that places this phosphate at the point of strand exchange. By contrast, corresponding modification of the proS oxygen destabilizes this conformation of the junction. Single-molecule analysis shows that both molecules are in a dynamic equilibrium between alternative stacking conformers, but the configuration of the methyl phosphonate determines the bias of the conformational equilibrium. It is likely that the stereochemical environment of the methyl group affects the interaction with metal ions in the center of the junction.

DNA↗

PubMed2004

Electrostatic interactions and the folding of the four-way DNA junction: analysis by selective methyl phosphonate substitution.

The structure and dynamics of the four-way (Holliday) junction are strongly dependent on the presence of metal ions. In this study, the importance of phosphate charge in and around the point of strand exchange has been explored by selective replacement with electrically neutral methyl phosphonate groups, guided by crystal structures of the junction in the folded, stacked X conformation. Junction conformation has been analysed by comparative gel electrophoresis and fluorescence resonance energy transfer (FRET). Three of sets of phosphate groups on the exchanging strands have been analysed; those at the point of strand exchange and those to their 3' and 5' sides. The exchanging and 3' phosphate groups form a box of negatively charged groups on the minor groove face of the junction, while the 5' phosphate groups face each other on the major groove side, with their proR oxygen atoms directed at one another. The largest effects are observed on substitution of the exchanging phosphate groups; replacement of both groups leads to the loss of the requirement for addition of metal ions to allow junction folding. When the equivalent phosphate groups on the continuous strands were substituted, a proportion of the junction folded into the alternative conformer so as to bring these phosphate groups onto the exchanging strands. These species did not interconvert, and thus this is likely to result from the alternative diasteromeric forms of the methyl phosphonate group. This shows that some of the conformational effects result from more than purely electrostatic interactions. Smaller but significant effects were observed on substitution of the flanking phosphate groups. All methyl phosphonate substitutions at these positions allowed folding to proceed at a reduced concentration of magnesium ions, with double substitutions more effective than single substitutions. Substitution of 5' phosphates resulted in a greater degree of folding at a given ionic concentration compared to the corresponding 3' phosphate substitutions. These results show that the phosphate groups at the point of strand exchange exert the largest electrostatic effect on junction folding, but a number of phosphate groups in the vicinity of the exchange region contribute to the overall effects.

DNA, Cruciform↗

PubMed2003

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 degrees 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.

2-Aminopurine↗

PubMed2003

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.

Base Sequence↗

PubMed2002

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.

Amino Acid Substitution↗