One of the disease-associated alleles identified contains two nonsynonymous polymorphisms, L85F/P786S, and the variant protein encoded by this allele shows impaired binding to MSH4. . This finding explains our recent atomic force microscopy study in which a dissociation of homologous HJs caused by migration over ≈100-bp region in the absence of Mg2+ cations observed as a fast event occurred between two consecutive scans (19). There is good evidence supporting this interpretation. An example of the slow hopping limit is shown in Fig. Domain 3 has a winged-helix fold and is the main domain that mediates nucleic acid binding [76]. The time dependence of FRET obtained in the presence of 10 mM Mg2+ (Fig. During genetic recombination, a Holliday junction is formed between the two strands of DNA and a double-stranded break in a DNA molecule leaves a 3’OH end exposed. 2b The segmented line is the same data without Gaussian noise. These arms may adopt one of several conformations depending on buffer salt concentrations and the sequence of nucleobases closest to the junction. A Holliday junction is a branched nucleic acid structure that contains four double-stranded arms joined together. The displaced strand in the extended ‘D-loop’ captures the second end by DNA strand annealing involving Rad52 (Figure 1, step 5a) to generate a dHJ by DNA synthesis (Step 6a). Significant increased usage of microhomology was also shown in junctions amplified from ex vivo splenic B cells in msh4−/− deficient cells (C57BL/6), although CSR in cells induced to switch in culture was identical to WT (Sekine et al., 2007). The DEE can be processed by disassembly through migration of the single ligated HJ to yield an NCO or through biased cleavage (at open arrowheads) by meiotic resolvases to generate a CO. It is hypothesized that through conformational changes, SisPINA can drive HJ branch migration. RuvC protein forms a dimeric complex with Holliday junctions (Figure 1A) assisted by RuvAB (not shown). The stability of the folded state determines how long the junction stalls. Hydrolysis of ATP through the MutSγ ATPase domains allows the MSH4–MSH5 heterodimers to form a sliding clamp that may displace the complex from the immediate vicinity of the dHJ, facilitating further clamp loading.262,263,264 In mice, MSH4 and MSH5 proteins localize in distinct foci along chromosome axes during zygonema of prophase I, usually in much higher numbers than the eventual number of COs in the nucleus, at around 150 per nucleus (Figure 1.7(I)). Intriguingly, different from other four ATPases, Sso0572 had unique characteristics. Red line (shifted for clarity, right axis) shows the same data but averaged as described in Methods. Domain 1 and domain 2 are RecA fold domain. 2b Rather, the junction migration over the large homology region was observed, suggesting that at favorable conditions one step can cover a large DNA region including several base pairs. It has long been recognized that the geometry of Holliday junctions is highly variable, and that this variability may be important for its functional role in biology [reviewed in Lilley (9), Lilley and White (10) and Hays et al. dHJ dissolution is mechanistically complex and requires the coordinate movement of both HJs in the dHJ toward each other by the BLM helicase (step 7c). 1998). Double-end engagement (DEE) model. Extensive branch migration over long stretches of DNA requires branch migration enzymes that provide a direction in an otherwise random walk. For example, the amino acids of Walker B motif of Sso0572 is different from others; Sso0572 is constantly expressed in the cell while the expression of the other four ATPases varied obviously. To analyze the effect of Mg cations on the branch migration rate the same samples were prepared in different concentrations of Mg2+ and spFRET experiments were performed. A Holliday junction is a branched nucleic acid structure that contains four double-stranded arms joined together. . However, progesterone has been shown to inhibit AID transcription and protein levels (Pauklin and Petersen-Mahrt, 2009). 9.3). On the other hand, if the diode is reverse biased, it blocks the electric current flow. , red line). Janet Stavnezer, ... Qiang Pan-Hammarström, in Advances in Immunology, 2010. As a rough estimate, the theoretical dependence of the FRET efficiency on the donor-acceptor distance shown in Fig. The drug was observed in clear electron density bound at the central crossover region (Fig. The most attractive candidate for this is the Holliday junction intermediate (HJ), the four-way DNA junction … The analysis of the data revealed the peaks in the FRET efficiency distribution correspond to peak values obtained for the data at 10 mM Mg2+ (see Supporting Text). ¶ To whom correspondence should be addressed. It can be formed both as a consequence of RecA/Rad51-catalysed strand invasion reactions, and by the reversal of impeded replication forks. In these determinations, the multiple crystal structures of four way junctions have identified some structural-effects of sequence and suggested an ACC trinucleotide pattern stabilizes Holliday junctions in a stacked conformation [11–13]. Theoretical Monte Carlo simulations of single-molecule FRET efficiencies of a six-site migrating HJ. DNA replication occurs in thre… (see also Fig. Correlated changes in the donor and acceptor fluorescence intensity are clearly seen. and its deviation δE. This allows a branch migration process to occur where the strands move through the junction point. That is, RuvAB and RuvC may act sequentially and without direct contact. Abbreviations: HJ, Holliday junction; spFRET, single-pair FRET. SMARCAL1 binds and branch-migrates Holliday junctions. RuvC produces either outcome. If the D-loop is dismantled, an NCO product is formed through SDSA. How the CO/NCO decision is made is not yet understood. This paper was submitted directly (Track II) to the PNAS office. The DNA substrates corresponding to each symbol and line color are the same in B and C. (D) Four-way branch migration substrate used in E–J. A candidate protein is Yen1 (human GEN1, Table 1), a nuclease that is capable of resolving Holliday junctions (HJs) with fully ligated strands (Matos et al., 2011). This finding is consistent with the recent publication from Ha's group (28) indicating that similar labeling arrangement is insensitive to changes of global junction conformation. This research was supported by National Institutes of Health Grant GM0062235 (to Y.L.L.). A wealth of structural information is available for immobile four-way junctions. [1] We use cookies to help provide and enhance our service and tailor content and ads. Figure 1. Intriguingly, in T. kodakaraensis Hjm can be knocked out, and the deleted mutant is slightly sensitive to UV and MMC [81]. A critical issue for this interpretation of the FRET data is that the dye-labeling method used is very sensitive to branch migration and much less sensitive to conformational transitions of the junction between unfolded and folded states (Fig. This observation is similar to the data obtained by Ha's group (14) for immobile HJs, suggesting that the mobile junction also flips between two conformations upon branch migration. According to the dHJ model, NCOs are formed primarily through SDSA. for a qualitative analysis of the branch migration data. In fact, at the beginning, Hjm was regarded as a candidate protein to promote HJ migration in P. furiosus [70]. In this model, up to half of all NCOs may arise from SDSA, giving rise to NCO products associated with hDNA in cis-orientation. Coaxial stacking is the tendency of nucleic acid blunt ends to bind to each other, by interactions between the exposed bases. This dependence predicts possible stepwise change of FRET intensities between 0.11 and 0.71. The hDNA tracts can then be molecularly analyzed to determine the orientation of the hDNA tracts, and through this, the structure of the recombination intermediate can be inferred. The crystal structures of two DNA four-way Holliday junctions complexed with a derivative of the covalently binding intercalating drug psoralen (Eichman et al., 2001), are remarkable demonstrations of the power of particular DNA sequences to form non-duplex structures. Subsequently, a gene encoding the protein which had the capacity to drive HJ migration was found by screening of a cosmid-based genomic library. . Holliday junction A Holliday junction is a branched nucleic acid structure that contains four double-stranded arms joined together. An answer to this question has come from the crystal structure of a Holliday junction with a bis-acridine molecule (Fig. A graphic illustration of this is the formation of a three-way DNA junction by an iron-containing molecule consisting of three bis-pyridylimine groups coordinated to two Fe2+ ions. We model the single-molecule FRET efficiency E(t) by using a stochastic Monte Carlo hopping process among N 2b One is the RuvA tetramer, which recognizes the structure of the Holliday junction. NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. After staying at the low FRET state (≈50 ms) the molecule adopts the state with a slightly higher FRET (≈0.38) and after 100 ms adopts the state with a very low FRET (≈0.15). Holiday junctions are also known as half chiasmas and Chi structures. The crystal structure (Oleksi et al., 2006) of a three-way junction involving, at the center of the structure a ligand formed by three bis-pyridylimine groups coordinated to two Fe2+ ions (shown as shaded spheres). Branch migration occurs while the junction is in an unfolded state and it stops when the molecule adopts a folded conformation. If RuvC has a sequence specificity for cleavage in living cells as it does in solution, this might explain genetic and biochemical evidence suggesting that RuvAB can facilitate RuvC activity on the junctions and enable its endonucleolytic cleavage function. The unexpected finding that four-way Holliday junctions can be formed by far shorter sequences than was hitherto envisaged, has led not only to study of the ligand complexes outlined above, but also to the realization that under some circumstances, ligand shape itself can control what type of DNA arrangement is formed. Biochemistry analysis showed that Hel308a in M. thermautotrophicus targets replication forks and unwinds lagging strand of a replication fork [69]. Genetic recombination occurs between homologous DNA molecules via a four-way (Holliday) junction intermediate. Seminal work with the human BLM-TOPOIIIα-RMI1 uncovered a novel mechanism to process dHJs by a mechanism termed dissolution (Wu and Hickson, 2003). Effect of Mg2+ Cations on the HJ Dynamic. (B) pBR322‐derived Holliday junctions used in this study (not to scale). Further, SisPINA can drive HJ branch migration and enhance cleavage activity on a fixed HJ by Hjc while the HJ branch migration activity of SisPINA can be inhibited by Hjc. 1c The challenge posed by this structure is whether selective molecules for junction structures can be devised from it, and if so, whether such ligands can have useful biological or therapeutic functions. SisPINA, a novel ATPase that is able to drive HJ migration was identified from S. islandicus [82]. Both MSH4 and MSH5 are critical for proper meiotic progression in the mouse, and the loss of either protein results in the absence of COs and complete sterility in males and females.265,266 In addition, they load with similar temporal and spatial frequency on synaptonemal complexes from human oocytes, indicating similar functions.268, The MutSγ heterodimer recruits a second MMR heterodimer, this time composed of the MutL homologs, MLH1 and MLH3 (MutLγ).269,270,271,272 Surprisingly, the complex is recruited sequentially in pachynema, MLH3 appearing on prophase I chromosome cores slightly prior to MLH1 in mouse, and localizing to cores even in Mlh1−/− mutants, indicating that MLH3 can bind to DNA without MLH1 present.273 MLH1 then proceeds to co-localize with MLH3 at all but a small subset of foci in mouse spermatocytes, thus marking the position of the Class I COs (Figure 1.7(J)).273 This Class I pathway is well characterized in lower eukaryotes, such as S. cerevisiae and A. thaliana, but very little is known about the control mechanisms in mammals.274,275 Studies of a well-characterized recombination hotspot in the mouse, Psmb9, as well as cytogenetic studies in mutants for Mlh1 and Mlh3 have indicated that 90–95% of COs depend upon the Class I, MLH1–MLH3-dependent, pathway, while the remaining COs are processed through a Class II pathway that is regulated by the MUS81–EME1 endonuclease (discussed further in this chapter).273,276,277,278 Additional proteins localize to sites of Class I COs in the mouse, and are crucial for proper progression of meiosis in the mouse. Results: RAD54 is a robust BM protein capable of bypassing extensive regions of DNA heterology. The estimated hopping rate is ≈100 μs, and in the presence of 10 mM Mg2+ folded states with the lifetimes as long as 100 ms were observed, suggesting that the rate of branch migration can drop 1,000-fold upon increase of the Mg2+ concentration. 2c These will then be able to form new junctions with any other homologous double helices they come across. (b) Synthetic Holliday junction analogs. From these data, Radford proposed the noncontinuous hDNA tracts resulted from a short-patch MMR system that was able to repair some mismatches in hDNA in concert with the canonical MMR machinery, and this short-patch MMR may include NER proteins, as shown in fission yeast. Product. Many proteins have been implicated in this role in both mammals and lower eukaryotes, including MUS81, MLH3, EXO1, YEN1, and SLX4; however, no definitive single resolvase has yet been discovered.283,284. The value 74 ± 5 ms calculated for a set of time trajectories similar to one shown in Fig. This is similar to the resolution in the single Holliday junction. At the same time, folding the junction is an effective brake that immediately blocks its movement. The crystal structure (Oleksi et al., 2006) of this complex bound to the simple hexanucleotide sequence d(CGTACG) shows that a three-way DNA junction has formed around it (Fig. This ancient and ubiquitous process is important for the repair of double-stranded breaks, the restart of stalled replication forks, and the creation of genetic diversity. Occurrence of Holliday junction-processing enzymes in eubacteria. However, the COs that were recovered with hDNA only appeared in one orientation. . 5b is very close to 71 ± 5.4 ms obtained for the design with alternate labeling (Table 1, which is published as supporting information on the PNAS web site). The protein was designated as Hjm for Holliday junction migration [70]. Holliday junction cleavage, which generates two unconnected DNA duplexes, is catalyzed by a group of structure-selective endonucleases called HJ resolvases. The variations of the FRET efficiency are considerably larger than statistical noise detected from an immobile junction (Fig. As in Fig. Figure modified from West (1997), from diagrams kindly provided by S.C. West. The role of the RMI2 cofactor in mammals remains to be determined. Holliday junction dynamics and branch migration: Single-molecule analysis. Hjm from P. furiosus can partially complement the defect of functions of recQ mutants in E. coli and Hjm functionally interacts with PCNA [71]. Alternatively, it is possible that RuvAB could make direct contact with RuvC to stimulate RuvC activity, though this is not needed in solution. ), suggesting that the observed FRET data dynamically follows the HJ undergoing branch migration. Four-way DNA intermediates, also known as Holliday junctions, ... whereas Yen1 was detected by screening a yeast gene fusion library for nucleases capable of Holliday junction resolution. It has the ability to catalyze strand separation in 3′–5′ direction. (a) Measured time dependence of the FRET efficiency in a 0.3 mM MgCl2 buffer (upper blue curve, left axis). These findings have led to a model whereby RecG targets the three-way junction at a D-loop created by 3′-end invasion (Fig. 2d The Holliday junction intermediate can move along the DNA to expand the newly paired heteroduplex region (branch migration). 12 are virtually identical (compare triangles and circles in Fig. However, these data cannot give the answer on the mechanism of branch migration, the major property of the Holliday junction. 2b 6a, which is published as supporting information on the PNAS web site. There is no null MSH5 mutation described in patients. The FRET efficiency time trajectories calculated from these data are shown in Fig. Thank you for your interest in spreading the word on PNAS. The Holliday junction (HJ), suggested in 1964 by Robin Holliday (), is a central intermediate in homologous and site-specific recombination ().Movement of the crossover junction along the DNA allowing for length extension of the heteroduplex is termed branch migration (Fig. However, when other msh5−/− mutants were analyzed on an MRL/Ipr or FVB background, although no CSR defect was observed in vitro, both Sμ–Sγ3 and Sμ–Sα junctions amplified from ex vivo splenic B cells showed significantly increased microhomology relative to WT littermates (Sekine et al., 2007). i values (Fig. 5b, reflecting the change in the donor-acceptor distance caused by branch migration during the time trajectories. If the D-loop is protected, both 3′ tails anneal to the same template and prime synthesis to form an unligated dHJ, termed DEE. 2 ways that the Holliday Junction is resolved(2 different planes) 1-without isomerization: breakage occurs in the same 2 DNA strands that were originally nicked at the beginning of this process→joining of strands→nonrecombinant chromosomes+heteroduplex region In contrast to Yen1/GEN1, the structure-selective endonuclease Mus81–Mms4 is very inefficient in cleaving HJs or dHJs when all strands are ligated, suggesting that it cleaves earlier intermediates before ligation (Ehmsen and Heyer, 2008). This prediction is in perfect agreement with papers (15, 16) in which the rate of branch migration was measured. DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. 2a Slx1–Slx4, another structure-selective nuclease, appears to play no obvious role in HR in budding yeast, based on the mutant phenotype, but has been implicated in HJ/dHJ processing in mammalian cells (Fricke and Brill, 2003; Castor et al., 2013). show no jumps of FRET values. This short sequence is expected to be present frequently in DNA. The DNA strands at the junction are held in an open configuration in the complex. The error bar on the right shows the time-averaged efficiency 〈E〉 Stephen Neidle, in Principles of Nucleic Acid Structure, 2008. Branch migration to the ‘left’ brings the complex to the sequence heterology formed by the single‐strand tails. Recombination occurs during … The results in Fig. Inset). 6b), suggesting that branch migration occurs through a selected number of discrete states along the branch migration path. This ancient and ubiquitous process is important for the repair of double-stranded breaks, the restart of stalled replication forks, and the creation of genetic diversity. Consequences of crossover: In most eukaryotes, a cell carries two copies of each gene, each referred to as an allele. Once formed, the four-way junction alone can undergo the stepwise exchange of base pairs known as spontaneous branch migration. The characteristic behavior of E(t) is quite different in the slow and rapid hopping limits. , blue line) are “noisy,” showing a statistical variation about a mean value. This rare long lifetime at lower Mg2+ concentration and the low rate of branch migration at the elevated 10 mM Mg2+ shows that Mg2+ produces an increase of the residence time of nonmigrating states of the junction. S. solfataricus (SsoHjm) [74], and P. furiosus (PfuHjm) [75] have been solved. The Holliday junctions in homologous recombination are between identical or nearly identical sequences, leading to a symmetric arrangement of sequences around the central junction. found that only half of the NCO synthesis tracts were associated with cis-hDNA. High conservation of meiotic proteins has led to an assumption that the dHJ model is also conserved across species; however, the model had never been directly tested in a metazoan because of an inability to reproduce a system for physical analysis of recombination intermediates like that in yeast. Regardless, COs and NCOs arising from the same intermediate sharply contrast the dHJ model. Figure 5.38. Holliday junctions are frequently used as the defining link motif in artificial supramolecular DNA assemblies (Seeman, 2005), although as yet ligand-directed assembly has not been seriously explored. However, the FRET variability is considerably less than observed for the same HJ design, but labeled differently (compare Fig. eliminated the canonical MMR machinery by mutating Msh6 [103]. In Drosophila meiosis, a Spo11-generated DSB is resected and one 3′ tail invades the homologous chromosome to form a D-loop. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism.