The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.
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ABSTRACT:
SUBMITTER: Rossi MJ
PROVIDER: S-EPMC3080997 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.
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Project description:In mammals, there are five Rad51 paralogs that form two distinct complexes in vivo. One complex is composed of Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and the other Rad51C-Xrcc3 (CX3). We co-expressed and purified human BCDX2 and CX3 protein complexes from insect cells and investigated their binding preferences and structure using transmission electron microscopy (TEM). We visualized the binding of BCDX2 and CX3 to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both complexes bind with exceptionally high specificity to the DNA junctions with little binding observed elsewhere on the DNAs. Further analysis of the structure of free or DNA-bound BCDX2 and CX3 complexes revealed a multimeric ring structure whose subunits are arranged into a flat disc around a central channel. This work provides the first EM visualization of BCDX2 and CX3 binding to Holliday junctions and forked DNAs and suggests the complexes form ring-shaped structures.
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Project description:The Holliday junction is a central intermediate in various genetic processes including homologous, site-specific recombination and DNA replication. Recent single molecule FRET experiments led to the model for branch migration as a stepwise stochastic process in which the branch migration hop is terminated by the folding of the junction. In this article, we studied the effect of the sequence on Holliday junction dynamics and branch migration process. We show that a GC pair placed at the border of the homologous region almost prevents the migration into this position. At the same time, insertion of a GC pair into the middle of the AT tract does not show this effect, however when the junction folds at this position, it resides at this position much longer time in comparison to the folding at AT pairs. Two contiguous GC pairs do not block migration as well and generally manifest the same effect as one GC pair--the junction when it folds resides at these positions for a relatively long time. The same elevated residence time was obtained for the design with the homology region that consists of only GC pairs. These data suggest a model for branch migration in which the sequence modulates the overall stochastic process of the junction dynamics and branch migration by the variability of the time that the junction dwells before making a migration hop.
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Project description:Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.
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Project description:DNA-PK (DNA-dependent protein kinase) is a double-strand break sensor involved in DNA repair and signal transduction. In the present study, we constructed site-directed cross-linking probes to explore the range of DNA discontinuities that are recognized by DNA-PK(CS) (DNA-PK catalytic subunit). A comparison between different substrate architectures showed that DNA-PK(CS) associates preferentially with the crossover region of synthetic Holliday junctions. This interaction with four-way junctions was preserved when biotin-streptavidin complexes were assembled at the termini to exclude the binding of Ku proteins. The association of DNA-PK(CS) with Holliday junctions was salt-labile even in the presence of Ku proteins, but this interaction could be stabilized when the DNA probes were incubated with the endogenous enzyme in nuclear extracts of human cells. Cross-linking of the endogenous enzyme in cellular extracts also demonstrated that DNA-PK(CS) binds to DNA ends and four-way junctions with similar affinities in the context of a nuclear protein environment. Kinase assays using p53 proteins as a substrate showed that, in association with four-way structures, DNA-PK(CS) adopts an active conformation different from that in the complex with linear DNA. Our results are consistent with a structure-specific, but Ku- and DNA end-independent, recruitment of DNA-PK(CS) to Holliday junction intermediates. This observation suggests an unexpected functional link between the two main pathways that are responsible for the repair of DNA double-strand breaks in mammalian cells.
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Project description:Repair of DNA double-strand breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumour suppression. However, despite its importance, the molecular intermediates of mitotic DSB repair remain undefined. The double Holliday junction (DHJ), presupposed to be the central intermediate for more than 25 years, has only been identified during meiotic recombination. Moreover, evidence has accumulated for alternative, DHJ-independent mechanisms, raising the possibility that DHJs are not formed during DSB repair in mitotically cycling cells. Here we identify intermediates of DSB repair by using a budding-yeast assay system designed to mimic physiological DSB repair. This system uses diploid cells and provides the possibility for allelic recombination either between sister chromatids or between homologues, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homologue joint molecule (JM) intermediates whose strand composition and size are identical to those of the canonical DHJ structures observed in meiosis. However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologues. Moreover, JMs seem to represent a minor pathway of DSB repair in mitotic cells, being detected at about tenfold lower levels (per DSB) than during meiotic recombination. Thus, although DHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.
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Project description:Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redβ. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redβ to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redβ-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA-protein nanostructures may find applications in the crystallization of DNA-protein complexes, our work shows the great potential of Redβ to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.
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Project description:Genetic recombination occurs between homologous DNA molecules via a four-way (Holliday) junction intermediate. 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. Once formed, the four-way junction alone can undergo the stepwise exchange of base pairs known as spontaneous branch migration. Conventional ensemble assays, useful for finding average migration rates over long sequences, have been unable to examine the affect of sequence and structure on the migration process. Here, we present a single-molecule spontaneous branch migration assay with single-base pair resolution in a study of individual DNA junctions that can undergo one step of migration. Junctions exhibit markedly different dynamics of exchange between stacking conformers depending on the point of strand exchange, allowing the moment at which branch migration occurs to be detected. The free energy landscape of spontaneous branch migration is found to be highly nonuniform and governed by two types of sequence-dependent barriers, with unmediated local migration being up to 10 times more rapid than the previously deduced average rate.
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Project description:In eukaryotes, RAD54 catalyzes branch migration (BM) of Holliday junctions, a basic process during DNA repair, replication, and recombination. RAD54 also stimulates RAD51 recombinase and has other activities. Here, we investigate the structural determinants for different RAD54 activities. We find that the RAD54 N-terminal domain (NTD) is responsible for initiation of BM through two coupled, but distinct steps; specific binding to Holliday junctions and RAD54 oligomerization. Furthermore, we find that the RAD54 oligomeric state can be controlled by NTD phosphorylation at S49, a CDK2 consensus site, which inhibits RAD54 oligomerization and, consequently, BM. Importantly, the effect of phosphorylation on RAD54 oligomerization is specific for BM, as it does not affect stimulation of RAD51 recombinase by RAD54. Thus, the transition of the oligomeric states provides an important control of the biological functions of RAD54 and, likely, other multifunctional proteins.
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