BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA.

Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individual's lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, replication protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins-of which BRCA2 is the defining member in humans-alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to-and the assembly of RAD51 filaments on-a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short preassembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation.

Top3α is required during the convergent migration step of double Holliday junction dissolution.

Although Blm and Top3α are known to form a minimal dissolvasome that can uniquely undo a double Holliday junction structure, the details of the mechanism remain unknown. It was originally suggested that Blm acts first to create a hemicatenane structure from branch migration of the junctions, followed by Top3α performing strand passage to decatenate the interlocking single strands. Recent evidence suggests that Top3α may also be important for assisting in the migration of the junctions. Using a mismatch-dHJ substrate (MM-DHJS) and eukaryotic Top1 (in place of Top3α), we show that the presence of a topoisomerase is required for Blm to substantially migrate a topologically constrained Holliday junction. When investigated by electron microscopy, these migrated structures did not resemble a hemicatenane. However, when Blm is together with Top3α, the dissolution reaction is processive with no pausing at a partially migrated structure. Potential mechanisms are discussed.

Improved methods for creating migratable Holliday junction substrates.

Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying late stage homologous recombination in vitro, construction of the substrate requires significant effort. In particular, there are three bottlenecks: (i) production of large quantities of single-stranded DNA; (ii) the loss of a significant portion of the DNA following the recombination step; and (iii) the loss of DNA owing to inefficient gel extraction. To address these limitations, we have made the following changes to the protocol: (i) use of a helper plasmid, rather than exogenous helper phage, to produce single-stranded DNA; (ii) use of the unidirectional C31 integrase system in place of the bidirectional Cre recombinase reaction; and (iii) gel extraction by DNA diffusion. Here, we describe the changes made to the materials and methods and characterize the substrates that can be produced, including migratable single Holliday junctions, hemicatenanes and a quadruple Holliday junction substrate.

Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.

Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured the nucleation and growth of RecA--and its eukaryotic homologue RAD51--on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5'→3' direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair, RecOR (RecO and RecR), accelerates both RecA nucleation and filament growth, and that the introduction of RecF further stimulates RecA nucleation.

Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes.

Genetic evidence indicates that Saccharomyces cerevisiae Sgs1, Top3, and Rmi1 resolve topologically linked intermediates arising from DNA replication and recombination. Using purified proteins, we show that Sgs1, Top3, Rmi1, and replication protein A (RPA) coordinate catenation and decatenation of dsDNA through sequential passage of single strands of DNA, establishing a unique pathway for dsDNA decatenation in eukaryotic cells. Sgs1 is required for dsDNA unwinding and, unexpectedly, also has a structural role in DNA strand passage. RPA promotes DNA unwinding by Sgs1 by trapping ssDNA, and it stimulates DNA strand passage by Top3. Paradoxically, Rmi1 has a unique regulatory capacity that slows DNA relaxation by Top3 but stimulates DNA decatenation. We establish that Rmi1 stabilizes the "open" Top3-DNA covalent complex formed as a transient intermediate of strand passage. This concerted activity of the Sgs1-Top3-Rmi1-RPA represents an important mechanism for disentangling structures resulting from the topological features of duplex DNA.

Essential functions of C terminus of Drosophila Topoisomerase IIIα in double holliday junction dissolution.

Topoisomerase IIIα (Top3α) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans, along with Blm and Rmi1/2. This important anti-recombinogenic function cannot be performed by Top3ß, the other type IA topoisomerase present in metazoans. The two share a catalytic core but diverge in their tail regions. To understand this difference in function, we investigated the role of the unique C terminus of Top3α. The Drosophila C terminus contains an insert region not conserved among metazoans. This insert contributes an independent interaction with Blm, which may account for the absence of Rmi1 in Drosophila. Mutant Top3α lacking this insert maintains the ability to perform dHJ dissolution but only partially rescues a top3α null fly line, indicating an in vivo role for the insert. Truncation of the C terminus has a minimal effect on the type IA relaxation activity of Top3α; however, dHJ dissolution is greatly reduced. The Top3α C terminus was found to strongly interact with both Blm and DNA, which are critical to the dissolution reaction; these interactions are greatly reduced in the truncated enzyme. The truncation mutant also cannot rescue the viability of top3α null flies, indicating an essential in vivo role. Our data therefore suggest that the Top3α C terminus has an important role in dHJ dissolution (by providing an interaction interface for Blm and DNA) and an essential function in vivo.

Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3.

A double Holliday junction (dHJ) is a central intermediate of homologous recombination that can be processed to yield crossover or non-crossover recombination products. To preserve genomic integrity, cells possess mechanisms to avoid crossing over. We show that Saccharomyces cerevisiae Sgs1 and Top3 proteins are sufficient to migrate and disentangle a dHJ to produce exclusively non-crossover recombination products, in a reaction termed "dissolution." We show that Rmi1 stimulates dHJ dissolution at low Sgs1-Top3 protein concentrations, although it has no effect on the initial rate of Holliday junction (HJ) migration. Rmi1 serves to stimulate DNA decatenation, removing the last linkages between the repaired and template DNA molecules. Dissolution of a dHJ is a highly efficient and concerted alternative to nucleolytic resolution that prevents crossing over of chromosomes during recombinational DNA repair in mitotic cells and thereby contributes to genomic integrity.

The phage T4 protein UvsW drives Holliday junction branch migration.

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.

Topoisomerase IIIalpha and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration.

It has long been suspected that a double Holliday junction (dHJ) could be resolved by a topoisomerase partnered with a helicase by convergent branch migration of the HJs. Genetic analysis of yeast TOP3 and SGS1 has lent considerable evidence to the notion that the protein products of these genes are involved in just such a process, although biochemical analysis of the metabolism of a dHJ has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a dHJ substrate that recapitulates many of the features of an endogenous dHJ and represents a much earlier intermediate in the resolution pathway. Here, we show that Drosophila topoisomerase IIIalpha (Topo IIIalpha) and Blm (a homolog of Sgs1) are capable of resolving this substrate to non-cross-over products and that this activity is stimulated by replication protein A (RPA). We investigated the ability of other Drosophila topoisomerases to perform this reaction in concert with Blm and RPA and discovered that this resolution activity is unique to Topo IIIalpha. Examination of the mechanism of resolution reveals that Topo IIIalpha, Blm, and RPA resolve this substrate by convergent migration of the two HJs toward each other, collapsing the dHJ. This mechanism stands in contrast to classic resolvase activities that use a structure-specific endonuclease to cleave the HJs.

A novel, topologically constrained DNA molecule containing a double Holliday junction: design, synthesis, and initial biochemical characterization.

The double Holliday junction (dHJ) is a central intermediate to homologous recombination, but biochemical analysis of the metabolism of this structure has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a novel dHJ substrate that consists of two small, double stranded DNA circles conjoined by two Holliday junctions (HJs). Its biochemical synthesis is based on the production of two pairs of single stranded circles from phagemids, followed by their sequential annealing with reverse gyrase. The sequence between the two HJs is identical on both strands, allowing the HJs to migrate without the generation of unpaired regions of DNA, whereas the distance between the HJs is on the order of gene conversion tracts thus far measured in Drosophila and mouse model systems. The structure of this substrate also provides similar topological constraint as would occur in an endogenous dHJ. Digestion of the dHJ substrate by T7 endonuclease I resolves the substrate into crossover and non-crossover products, as predicted by the Szostak model of double strand break repair. This substrate will greatly facilitate the examination of the mechanism of resolution of double Holliday junctions.

Reverse gyrase functions as a DNA renaturase: annealing of complementary single-stranded circles and positive supercoiling of a bubble substrate.

Reverse gyrase is a hyperthermophile-specific enzyme that can positively supercoil DNA concomitant with ATP hydrolysis. However, the DNA supercoiling activity is inefficient and requires an excess amount of enzyme relative to DNA. We report here several activities that reverse gyrase can efficiently mediate with a substoichiometric amount of enzyme. In the presence of a nucleotide cofactor, reverse gyrase can readily relax negative supercoils, but not the positive ones, from a plasmid DNA substrate. Reverse gyrase can completely relax positively supercoiled DNA, provided that the DNA substrate contains a single-stranded bubble. Reverse gyrase efficiently anneals complementary single-stranded circles. A substoichiometric amount of reverse gyrase can insert positive supercoils into DNA with a single-stranded bubble, in contrast to plasmid DNA substrate. We have designed a novel method based on phage-mid DNA vectors to prepare a circular DNA substrate containing a single-stranded bubble with defined length and sequence. With these bubble DNA substrates, we demonstrated that efficient positive supercoiling by reverse gyrase requires a bubble size larger than 20 nucleotides. The activities of annealing single-stranded DNA circles and positive supercoiling of bubble substrate demonstrate that reverse gyrase can function as a DNA renaturase. These biochemical activities also suggest that reverse gyrase can have an important biological function in sensing and eliminating unpaired regions in the genome of a hyperthermophilic organism.

Drosophila melanogaster topoisomerase IIIalpha preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development.

Eukaryotic type IA topoisomerases are important for the normal function of the cell, and in some cases essential for the organism, although their role in DNA metabolism remains to be elucidated. In this study, we cloned Drosophila melanogaster topoisomerase (topo) IIIalpha from an embryonic cDNA library and expressed and purified the protein to >95% homogeneity. This enzyme partially relaxes a hypernegatively supercoiled plasmid substrate consistent with other purified topo IIIs. A novel, covalently closed bubble substrate was prepared for this study, which topo IIIalpha fully relaxed, regardless of the handedness of the supercoils. Experiments with the bubble substrate demonstrate that topo IIIalpha has much different reaction preferences from those obtained by plasmid substrate-based assays. This is presumably due to the fact that solution conditions can affect the structure of plasmid based substrates and therefore their suitability as a substrate. A mutant allele of the Top3alpha gene, Top3alpha191, was isolated through imprecise excision mutagenesis of an existing P-element inserted in the first intron of the gene. Top3alpha191 is recessive lethal, with most of the homozygous individuals surviving to pupation but never emerging to adulthood. Whereas this mutation can be rescued by a Top3alpha transgene, ubiquitous overexpression of D. melanogaster topo IIIbeta cannot rescue this allele.