Four-way Holliday Junction Research Articles

Bacteriophage lambda site-specific recombination.

The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage-encoded Int and Xis proteins and the host-encoded integration host factor and factor for inversion stimulation proteins. Structures of higher-order protein-DNA complexes of the four-way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site-specific recombination, with an emphasis on key studies completed over the last decade.

Analysis of non-canonical three- and four-way DNA junctions.

The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds-a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively-were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (Kd and EC50) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.

Conformational dynamics and mechanical properties of biomimetic RNA, DNA, and RNA-DNA hybrid nanotubes: an atomistic molecular dynamics study.

With the nanotechnology boom, artificially designed nucleic acid nanotubes have aroused interest due to their practical applications in nanorobotics, vaccine design, membrane channels, drug delivery, and force sensing. In this paper, computational study was performed to investigate the structural dynamics and mechanical properties of RNA nanotubes (RNTs), DNA nanotubes (DNTs), and RNA-DNA hybrid nanotubes (RDHNTs). So far, the structural and mechanical properties of RDHNTs have not been examined in experiments or theoretical calculations, and there is limited knowledge regarding these properties for RNTs. Here, the simulations were carried out using the equilibrium molecular dynamics (MD) and steered molecular dynamics (SMD) approaches. Using in-house scripting, we modeled hexagonal nanotubes composed of six double-stranded molecules connected by four-way Holliday junctions. Classical MD analyses were performed on the collected trajectory data to investigate structural properties. Analyses of the microscopic structural parameters of RDHNT indicated a structural transition from the A-form to a conformation between the A- and B-forms, which may be attributable to the increased rigidity of RNA scaffolds compared to DNA staples. Comprehensive research on the elastic mechanical properties was also conducted based on spontaneous thermal fluctuations of nanotubes and employing the equipartition theorem. The Young's modulus of RDHNT (E = 165 MPa) and RNT (E = 144 MPa) was found to be almost the same and nearly half of that found for DNT (E = 325 MPa). Furthermore, the results showed that RNT was more resistant to bending, torsional, and volumetric deformations than DNT and RDHNT. We also used non-equilibrium SMD simulations to acquire comprehensive knowledge of the mechanical response of nanotubes to tensile stress.

Finding and cleaving a holliday junction in long double-stranded DNA: a complete resolution reaction trajectory by single-molecule tracking

Resolution of four-way Holliday junctions (HJs) is a critical intermediate step of Homologous Recombination. HJs are processed by abundant junction-resolving endonucleases that introduce two hydrolytic cleavages. Although their catalytic activity and interaction mode with HJ-DNA have been characterized in great detail by biochemical and structural studies, it remains unclear how the endonucleases find their substrate located in kilobase pairs of duplex DNA. Here, we studied the interaction of the T7 endonuclease I with a long dsDNA molecule that has a junction located at its center.

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1.

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule Förster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Resolution of the Holliday junction recombination intermediate by human GEN1 at the single-molecule level.

Human GEN1 is a cytosolic homologous recombination protein that resolves persisting four-way Holliday junctions (HJ) after the dissolution of the nuclear membrane. GEN1 dimerization has been suggested to play key role in the resolution of the HJ, but the kinetic details of its reaction remained elusive. Here, single-molecule FRET shows how human GEN1 binds the HJ and always ensures its resolution within the lifetime of the GEN1-HJ complex. GEN1 monomer generally follows the isomer bias of the HJ in its initial binding and subsequently distorts it for catalysis. GEN1 monomer remains tightly bound with no apparent dissociation until GEN1 dimer is formed and the HJ is fully resolved. Fast on- and slow off-rates of GEN1 dimer and its increased affinity to the singly-cleaved HJ enforce the forward reaction. Furthermore, GEN1 monomer binds singly-cleaved HJ tighter than intact HJ providing a fail-safe mechanism if GEN1 dimer or one of its monomers dissociates after the first cleavage. The tight binding of GEN1 monomer to intact- and singly-cleaved HJ empowers it as the last resort to process HJs that escape the primary mechanisms.

Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes.

Four-way Holliday junctions in DNA are the central intermediates of genetic recombination and must be processed into regular duplex species. One mechanism for achieving this is called resolution, brought about by structure-selective nucleases. GEN1 is an important junction-resolving enzyme in eukaryotic cells, a member of the FEN1/EXO1 superfamily of nucleases. While human GEN1 is difficult to work with because of aggregation, orthologs from thermophilic fungi have been identified using bioinformatics and have proved to have excellent properties. Here, the expression and purification of this enzyme from Chaetomium thermophilum is described, together with the means of investigating its biochemical properties. The enzyme is quite similar to junction-resolving enzymes from lower organisms, binding to junctions in dimeric form, introducing symmetrical bilateral cleavages, the second of which is accelerated to promote productive resolution. Crystallization of C. thermophilum GEN1 is described, and the structure of a DNA-product complex. Juxtaposition of complexes in the crystal lattice suggests how the structure of a dimeric enzyme with an intact junction is organized.

TRF2 binds branched DNA to safeguard telomere integrity.

Although t-loops protect telomeres, they are at risk of cleavage by Holliday junction (HJ) resolvases if branch migration converts the three-way t-loop junction into four-way HJs. T-loop cleavage is repressed by the TRF2 basic domain, which binds three- and four-way junctions and protects HJs in vitro. By replacing the basic domain with bacterial-protein domains binding three- and four-way junctions, we demonstrated the in vivo relevance of branched-DNA binding. Branched-DNA binding also repressed PARP1, presumably by masking the PARP1 site in the t-loop junction. Although PARP1 recruits HJ resolvases and promotes t-loop cleavage, PARP1 activation alone did not result in t-loop cleavage, thus suggesting that the basic domain also prevents formation of HJs. Concordantly, removal of HJs by BLM helicase mitigated t-loop cleavage in response to loss of the basic domain. We propose that TRF2 masks and stabilizes the t-loop three-way junction, thereby protecting telomeres from detrimental deletions and PARP1 activation.

Resolution of single and double Holliday junction recombination intermediates by GEN1

Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicase-TopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5'-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase, Escherichia coli RuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5'-A/TTTG/C-3'.

Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1

In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg2+ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg2+ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.

Holliday Junction Thermodynamics and Structure: Comparisons of Coarse-Grained Simulations and Experiments

The DNA Holliday junction plays a vital role in genetic recombination and DNA repair. All-atom simulations of DNA junctions have proved challenging, owing to the substantial size and time scales required to examine conformational changes or junction melting. Here, we combine simulations and experiments to evaluate the possibility of using the 3SPN.2 model, a coarse-grained molecular representation designed to mimic B-DNA, to reproduce the properties of a DNA four-way Holliday junction. We find that the model reproduces our experimental measurements of the junction melting temperature dependence on salt concentration to within 3%. The model reproduces the bias between open and stacked conformations, as well as the relative population of stacked isomers at high salt concentration. We further predict the population of the iso-I and iso-II forms is nearly independent of salt concentration. We directly observe proposed tetrahedral intermediate sub-states implicated in conformational transitions, suggesting that these conformations, rather than planar open states, facilitate these transitions. Finally, we compare our results for inter-duplex angle (IDA) with previous fluorescence experiments and all-atom calculations based on the AMBER force field. We find a close correspondence between the average IDA of our all-atom and coarse-grained simulations, but find that the “arms” of the junction in the 3SPN.2 model exhibit larger fluctuations, apparently due to the implicit nature of solvent and ion interactions. Our findings demonstrate that the 3SPN.2 model captures junction properties that are not presently accessible to all-atom studies, opening the possibilities to simulate complex aspects of junction behavior.

Molecular dynamics simulation of hydrated d(CGGGTACCCG)4 as a four-way DNA Holliday junction and comparison with the crystallographic structure

Molecular dynamics (MD) simulation of decamer sequence (CGGGTACCCG)4 as a four-way Holliday junction is reported here for 15.0 ns at three different temperatures 100, 200 and 300 K, respectively, using AMBER force field. Particle mesh Ewald method has been utilised to deal long-range interaction potentials. After MD simulation, various parameters of the junction model including backbone and helical parameters have been worked out and the dynamical pathway is discussed. Structural analysis and geometrical calculations were carried out through X3DNA. The computational results obtained are compared with the previously reported crystallographic outcomes. The width and depth of the major and minor grooves of the duplex of the four arms of the DNA junction have been calculated. The variations in the C1′–C1′ distances between the two complementary strands are discussed in detail. A close observation of the results reveals that the conformation of the average simulated structure at low temperature is of ‘B’ form and the structural integrity of the DNA junction having a twofold sequence symmetry is temperature dependent. It also seems that besides the other parameters (i.e. presence of ions, solvents, etc.), temperature may be playing a key role in preserving the structural integrity of the DNA junction.

Direct observation of stalled fork restart via fork regression in the T4 replication system.

The restart of a stalled replication fork is a major challenge for DNA replication. Depending on the nature of the damage, different repair processes might be triggered; one is template switching, which is a bypass of a leading-strand lesion via fork regression. Using magnetic tweezers to study the T4 bacteriophage enzymes, we have reproduced in vitro the complete process of template switching. We show that the UvsW DNA helicase in cooperation with the T4 holoenzyme can overcome leading-strand lesion damage by a pseudostochastic process, periodically forming and migrating a four-way Holliday junction. The initiation of the repair process requires partial replisome disassembly via the departure of the replicative helicase. The results support the role of fork regression pathways in DNA repair.

SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication

SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1) maintains genome integrity during DNA replication. Here we investigated its mechanism of action. We found that SMARCAL1 travels with elongating replication forks, and its absence leads to MUS81-dependent double-strand break formation. Binding to specific nucleic acid substrates activates SMARCAL1 activity in a reaction that requires its HARP2 (Hep-A-related protein 2) domain. Homology modeling indicates that the HARP domain is similar in structure to the DNA-binding domain of the PUR proteins. Limited proteolysis, small-angle X-ray scattering, and functional assays indicate that the core enzymatic unit consists of the HARP2 and ATPase domains that fold into a stable structure. Surprisingly, SMARCAL1 is capable of binding three-way and four-way Holliday junctions and model replication forks that lack a designed ssDNA region. Furthermore, SMARCAL1 remodels these DNA substrates by promoting branch migration and fork regression. SMARCAL1 mutations that cause Schimke immunoosseous dysplasia or that inactivate the HARP2 domain abrogate these activities. These results suggest that SMARCAL1 continuously surveys replication forks for damage. If damage is present, it remodels the fork to promote repair and restart. Failures in the process lead to activation of an alternative repair mechanism that depends on MUS81-catalyzed cleavage of the damaged fork.

Structure of d(CGGGTACCCG)4 as a four-way Holliday junction.

The crystal structure of the decamer sequence d(CGGGTACCCG)(4) as a four-way Holliday junction has been determined at 2.35 Å resolution. The sequence was designed in order to understand the principles that govern the relationship between sequence and branching structure. It crystallized as a four-way junction structure with an overall geometry similar to those of previously determined Holliday junction structures.

Binding and Bending Parameters of Integration Host Factor to Four-Way Holliday Junction

Binding and Bending Parameters of Integration Host Factor to Four-Way Holliday JunctionIntegration host factor (IHF) is a small heterodimeric protein that sequence specifically binds the minor groove of DNA and facilitates a bend of nearly 180 degrees. This bending is crucial for cellular processes such as recombination, replication and transcription. Previous work in the Mukerji lab characterized the binding properties of the structurally similar but sequence non-specific DNA-binding protein HU, to duplex and Holliday junction DNA. This research demonstrated that HU binds to the central region of the junction with nanomolar affinity and prefers the stacked form of the Holliday junction. Given the similarities in structure and function of HU and IHF, we elected to study the binding capability of IHF to the Holliday junction. We have compared the binding affinity of IHF for Holliday junction DNA with its binding affinity to duplex DNA with and without a consensus sequence. Binding measurements were performed using florescence intensity and anisotropy methods and confirmed with the gel mobility shift assay. All binding assays established that IHF binds to the Holliday junction lacking a consensus sequence with high affinity (∼3 nanomolar Kd) similar to HU, suggesting that both proteins might recognize and bind similar structural aspects of the Holliday junction. Moreover, anisotropy affinity measurements demonstrated that IHF binds the Holliday junction with similar affinity as it does to duplex DNA containing its consensus sequence. The span of binding capabilities exhibited by IHF indicate that it can function both as a specific and non-sequence specific DNA binding protein.

The Werner Syndrome Protein Binds Replication Fork and Holliday Junction DNAs as an Oligomer

Werner syndrome is an inherited disease displaying a premature aging phenotype. The gene mutated in Werner syndrome encodes both a 3' --> 5' DNA helicase and a 3' --> 5' DNA exonuclease. Both WRN helicase and exonuclease preferentially utilize DNA substrates containing alternate secondary structures. By virtue of its ability to resolve such DNA structures, WRN is postulated to prevent the stalling and collapse of replication forks that encounter damaged DNA. Using electron microscopy, we visualized the binding of full-length WRN to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both wild-type WRN and a helicase-defective mutant bind with exceptionally high specificity (>1000-fold) to DNA secondary structures at the replication fork and at Holliday junctions. Little or no binding is observed elsewhere on the DNA molecules. Calculations of the molecular weight of full-length WRN revealed that, in solution, WRN exists predominantly as a dimer. However, WRN bound to DNA is larger; the mass is consistent with that of a tetramer.

Ligand Bridging of the DNA Holliday Junction: Molecular Recognition of a Stacked‐X Four‐Way Junction by a Small Molecule

Planning a Holliday: A new mode of binding to a stacked-X, four-way Holliday junction is described in which a chromophore molecule binds across the center of the junction and two adenine residues are replaced by the acridine chromophores at either side of the crossover. This binding mode is specific for the Holliday junction and does not cause unwinding of the DNA helices.

PROSIT, AN ONLINE SERVICE TO CALCULATE PSEUDOROTATIONAL PARAMETERS OF NUCLEOSIDES AND NUCLEOTIDES

The online service PROSIT (Pseudo-Rotational Online Service and Interactive Tool) is a free service available at http://cactus.nci.nih.gov/prosit/ that performs pseudorotational analysis of nucleosides(tides). PROSIT reads the 3D coordinates of nucleosides and returns the pseudorotational phase angle P, puckering amplitude νmax, and other related information. As examples, the sugar conformations in a parallel-stranded guanine tetraplex and a four-way Holliday junction are presented here.

Molecular modeling of the chromatosome particle.

In an effort to understand the role of the linker histone in chromatin folding, its structure and location in the nucleosome has been studied by molecular modeling methods. The structure of the globular domain of the rat histone H1d, a highly conserved part of the linker histone, built by homology modeling methods, revealed a three-helical bundle fold that could be described as a helix-turn-helix variant with its characteristic properties of binding to DNA at the major groove. Using the information of its preferential binding to four-way Holliday junction (HJ) DNA, a model of the domain complexed to HJ was built, which was subsequently used to position the globular domain onto the nucleosome. The model revealed that the primary binding site of the domain interacts with the extra 20 bp of DNA of the entering duplex at the major groove while the secondary binding site interacts with the minor groove of the central gyre of the DNA superhelix of the nucleosomal core. The positioning of the globular domain served as an anchor to locate the C-terminal domain onto the nucleosome to obtain the structure of the chromatosome particle. The resulting structure had a stem-like appearance, resembling that observed by electron microscopic studies. The C-terminal domain which adopts a high mobility group (HMG)-box-like fold, has the ability to bend DNA, causing DNA condensation or compaction. It was observed that the three S/TPKK motifs in the C-terminal domain interact with the exiting duplex, thus defining the path of linker DNA in the chromatin fiber. This study has provided an insight into the probable individual roles of globular and the C-terminal domains of histone H1 in chromatin organization.