Real-time measurements of the nucleation, growth and dissociation of single Rad51-DNA nucleoprotein filaments

Human Rad51 (hRad51), the protein central to DNA pairing and strand exchange during homologous recombination, polymerizes on DNA to form nucleoprotein filaments. By making use of magnetic tweezers to manipulate individual DNA molecules, we measured the nucleation and growth of hRad51 nucleoprotein filaments, and their subsequent disassembly in real time. The dependence of the initial polymerization rate upon the concentration of hRad51 suggests that the rate-limiting step is the formation of a nucleus involving 5.5 ± 1.5 hRad51 monomers, corresponding to one helical turn of the hRad51 nucleoprotein filament. Polymerization is highly cooperative (i.e. a nucleation-limited reaction) at low concentrations and less cooperative (a growth-limited reaction) at high concentrations of the protein. We show that the observed preference of hRad51 to form nucleoprotein filaments on double-stranded DNA rather than on single-stranded DNA is due to the fact that it depolymerizes much faster from ssDNA than from dsDNA: indeed, hRad51 polymerizes faster on ssDNA than on dsDNA. Hydrolysis of ATP by hRad51 does not correlate with its dissociation from dsDNA. This suggests that hRad51 does not depolymerize rapidly from dsDNA after strand exchange but stays bound to the heteroduplex, highlighting the importance of partner proteins to facilitate hRad51 depolymerization from dsDNA.     

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.     

FBH1 Helicase Disrupts RAD51 Filaments in Vitro and Modulates Homologous Recombination in Mammalian Cells

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.     

Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase

Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins.     

Visualizing RAD51-mediated joint molecules: implications for recombination mechanism and the effect of sequence heterology

The defining event in homologous recombination is the exchange of base-paired partners between a single-stranded (ss) DNA and a homologous duplex driven by recombinase proteins, such as human RAD51. To understand the mechanism of this essential genome maintenance event, we analyzed the structure of RAD51–DNA complexes representing strand exchange intermediates at nanometer resolution by scanning force microscopy. Joint molecules were formed between substrates with a defined ssDNA segment and homologous region on a double-stranded (ds) partner. We discovered and quantified several notable architectural features of RAD51 joint molecules. Each end of the RAD51-bound joints had a distinct structure. Using linear substrates, a 10-nt region of mispaired bases blocked extension of joint molecules in all examples observed, whereas 4 nt of heterology only partially blocked joint molecule extension. Joint molecules, including 10 nt of heterology, had paired DNA on either side of the heterologous substitution, indicating that pairing could initiate from the free 3′end of ssDNA or from a region adjacent to the ss–ds junction. RAD51 filaments covering joint ss–dsDNA regions were more stable to disassembly than filaments covering dsDNA. We discuss how distinct structural features of RAD51-bound DNA joints can play important roles as recognition sites for proteins that facilitate and control strand exchange.     

Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules

The human DNA repair protein RAD51 is the crucial component of helical nucleoprotein filaments that drive homologous recombination. The molecular mechanistic details of how this structure facilitates the requisite DNA strand rearrangements are not known but must involve dynamic interactions between RAD51 and DNA. Here, we report the real-time kinetics of human RAD51 filament assembly and disassembly on individual molecules of both single- and double-stranded DNA, as measured using magnetic tweezers. The relative rates of nucleation and filament extension are such that the observed filament formation consists of multiple nucleation events that are in competition with each other. For varying concentration of RAD51, a Hill coefficient of 4.3 ± 0.5 is obtained for both nucleation and filament extension, indicating binding to dsDNA with a binding unit consisting of multiple (≥4) RAD51 monomers. We report Monte Carlo simulations that fit the (dis)assembly data very well. The results show that, surprisingly, human RAD51 does not form long continuous filaments on DNA. Instead each nucleoprotein filament consists of a string of many small filament patches that are only a few tens of monomers long. The high flexibility and dynamic nature of this arrangement is likely to facilitate strand exchange.     

A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated strand exchange

Human BRCA2, a breast and ovarian cancer suppressor, binds to the DNA recombinase RAD51 through eight conserved BRC repeats, motifs of approximately 30 residues, dispersed across a large region of the protein. BRCA2 is essential for homologous recombination in vivo, but isolated BRC repeat peptides can prevent the assembly of RAD51 into active nucleoprotein filaments in vitro, suggesting a model in which BRCA2 sequesters RAD51 in undamaged cells, and promotes recombinase function after DNA damage. How BRCA2 might fulfill these dual functions is unclear. We have purified a fragment of human BRCA2 (BRCA2(BRC1-8)) with 1127 residues spanning all 8 BRC repeats but excluding the C-terminal DNA-binding domain (BRCA2(CTD)). BRCA2(BRC1-8) binds RAD51 nucleoprotein filaments in a ternary complex, indicating it may organize RAD51 on DNA. Human RAD51 is relatively ineffective in vitro at strand exchange between homologous DNA molecules unless non-physiological ions like NH4+ are present. In an ionic milieu more typical of the mammalian nucleus, BRCA2(BRCI-8) stimulates RAD51-mediated strand exchange, suggesting it may be an essential co-factor in vivo. Thus, the human BRC repeats, embedded within their surronding sequences as an eight-repeat unit, mediate homologous recombination independent of the BRCA2(CTD) through a previously unrecognized role in control of RAD51 activity.     

The differential extension in dsDNA bound to Rad51 filaments may play important roles in homology recognition and strand exchange

RecA and Rad51 proteins play an important role in DNA repair and homologous recombination. For RecA, X-ray structure information and single molecule force experiments have indicated that the differential extension between the complementary strand and its Watson–Crick pairing partners promotes the rapid unbinding of non-homologous dsDNA and drives strand exchange forward for homologous dsDNA. In this work we find that both effects are also present in Rad51 protein. In particular, pulling on the opposite termini (3′ and 5′) of one of the two DNA strands in a dsDNA molecule allows dsDNA to extend along non-homologous Rad51-ssDNA filaments and remain stably bound in the extended state, but pulling on the 3′5′ ends of the complementary strand reduces the strand-exchange rate for homologous filaments. Thus, the results suggest that differential extension is also present in dsDNA bound to Rad51. The differential extension promotes rapid recognition by driving the swift unbinding of dsDNA from non-homologous Rad51-ssDNA filaments, while at the same time, reducing base pair tension due to the transfer of the Watson–Crick pairing of the complementary strand bases from the highly extended outgoing strand to the slightly less extended incoming strand, which drives strand exchange forward.     

Tyrosine phosphorylation enhances RAD52-mediated annealing by modulating its DNA binding

RAD52 protein has an important role in homology-directed DNA repair by mediating RAD51 nucleoprotein filament formation on single-stranded DNA (ssDNA) protected by replication protein-A (RPA) and annealing of RPA-coated ssDNA. In human, cellular response to DNA damage includes phosphorylation of RAD52 by c-ABL kinase at tyrosine 104. To address how this phosphorylation modulates RAD52 function, we used an amber suppressor technology to substitute tyrosine 104 with chemically stable phosphotyrosine analogue (p-Carboxymethyl-L-phenylalanine, pCMF). The RAD52(Y104pCMF) retained ssDNA-binding activity characteristic of unmodified RAD52 but showed lower affinity for double-stranded DNA (dsDNA) binding. Single-molecule analyses revealed that RAD52(Y104pCMF) specifically targets and wraps ssDNA. While RAD52(Y104pCMF) is confined to ssDNA region, unmodified RAD52 readily diffuses into dsDNA region. The Y104pCMF substitution also increased the ssDNA annealing rate and allowed overcoming the inhibitory effect of dsDNA. We propose that phosphorylation at Y104 enhances ssDNA annealing activity of RAD52 by attenuating dsDNA binding. Implications of phosphorylation-mediated activation of RAD52 annealing activity are discussed.     

Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein

The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1–single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.     

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments.     

Context-Dependent Remodeling of Rad51–DNA Complexes by Srs2 Is Mediated by a Specific Protein–Protein Interaction

The yeast Srs2 helicase removes Rad51 nucleoprotein filaments from single-stranded DNA (ssDNA), preventing DNA strand invasion and exchange by homologous recombination. This activity requires a physical interaction between Srs2 and Rad51, which stimulates ATP turnover in the Rad51 nucleoprotein filament and causes dissociation of Rad51 from ssDNA. Srs2 also possesses a DNA unwinding activity and here we show that assembly of more than one Srs2 molecule on the 3′ ssDNA overhang is required to initiate DNA unwinding. When Rad51 is bound on the double-stranded DNA, its interaction with Srs2 blocks the helicase (DNA unwinding) activity of Srs2. Thus, in different DNA contexts, the physical interaction of Rad51 with Srs2 can either stimulate or inhibit the remodeling functions of Srs2, providing a means for tailoring DNA strand exchange activities to enhance the fidelity of recombination.     

RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.     

RAD51 variant proteins from human lung and kidney tumors exhibit DNA strand exchange defects

In human cells, error-free repair of DNA double-strand breaks requires the DNA pairing and strand exchange activities of RAD51 recombinase. Activation of RAD51 recombination activities requires the assembly of RAD51 presynaptic filaments on the single-stranded DNA that forms at resected DSB ends. Mutations in proteins that control presynaptic filament assembly, such as BRCA2, and in RAD51 itself, are associated with human breast cancer. Here we describe the properties of two mutations in RAD51 protein that derive from human lung and kidney tumors, respectively. Sequence variants Q268P and Q272L both map to the DNA binding loop 2 (L2) region of RAD51, a motif that is involved in DNA binding and in the allosteric activation of ATP hydrolysis and DNA strand exchange activities. Both mutations alter the thermal stability, DNA binding, and ATPase properties of RAD51, however both variants retain intrinsic DNA strand exchange activity towards oligonucleotide substrates under optimized conditions. In contrast, both Q268P and Q272L variants exhibit drastically reduced DNA strand exchange activity in reaction mixtures containing long homologous ssDNA and dsDNA substrates and human RPA protein. Mixtures of wild-type and variant proteins also exhibit reduced DNA strand exchange activity, suggesting that heterozygous mutations could negatively affect DNA recombination and repair processes in vivo. Together, the findings of this study suggest that hypomorphic missense mutations in RAD51 protein could be drivers of genomic instability in cancer cells, and thereby contribute to the etiology of metastatic disease.     

Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament

Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.     

Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex

Saccharomyces cerevisiae Dmc1, a meiosis-specific homologue of RecA, catalyzes homologous pairing and strand exchange during meiotic DNA recombination. The purified budding yeast Dmc1 (ScDmc1) protein exhibits much weaker recombinase activity in vitro as compared to that of the Escherichia coli RecA protein. Using atomic force microscopy (AFM) with carbon nanotube tips, we found ScDmc1 forms rings with an external diameter of 18 nm and a central cavity of 4 nm. In the presence of single-stranded DNA (ssDNA), the majority of the ScDmc1 protein (90%) bound DNA as protein rings; only a small faction (10%) was able to form filamentous structure. In contrast, nearly all RecA proteins form fine helical nucleoprotein filaments with ssDNA under identical conditions. RecA-mediated recombinase activity is initiated through the nucleation of RecA onto ssDNA to form helical nucleoprotein filaments. Our results support the notion that ScDmc1 becomes catalytically active only when it forms a helical nucleoprotein filament with ssDNA.     

Biochemical characterization of the human RAD51 protein. III. Modulation of DNA binding by adenosine nucleotides

Adenosine nucleotides affect the ability of RecA small middle dotsingle-stranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATPgammaS affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATPgammaS, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51 small middle dotssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA small middle dotssDNA binding form. The available evidence suggests that a low molecular weight hRAD51 small middle dotssDNA binding form (hRAD51 small middle dotssDNA(low)) correlates with active ADP and ATP processing. A high molecular weight hRAD51 small middle dotssDNA aggregate (hRAD51 small middle dotssDNA(high)) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: BRCA2: One Small Step for DNA Repair,One Giant Protein Purified

DNA damage, malfunctions in DNA repair, and genomic instability are processes that intersect at the crossroads of carcinogenesis. Underscoring the importance of DNA repair in breast and ovarian tumorigenesis is the familial inherited cancer predisposition gene BRCA2. The role of BRCA2 in DNA double-strand break repair was first revealed based on its interaction with RAD51, a central player in homologous recombination. The RAD51 protein forms a nucleoprotein filament on single-stranded DNA, invades a DNA duplex, and initiates a search for homology. Once a homologous DNA sequence is found, the DNA is used as a template for the high-fidelity repair of the DNA break. Many of the biochemical features that allow BRCA2 to choreograph the activities of RAD51 have been elucidated and include: targeting RAD51 to single-stranded DNA while inhibiting binding to dsDNA, reducing the ATPase activity of RAD51, and facilitating the displacement of the single-strand DNA binding protein, Replication Protein A. These reinforcing activities of BRCA2 culminate in the correct positioning of RAD51 onto a processed DNA double-strand break and initiate its faithful repair by homologous recombination. In this review, I will address current biochemical data concerning the BRCA2 protein and highlight unanswered questions regarding BRCA2 function in homologous recombination and cancer.     

Real-time solution measurement of RAD51- and RecA-mediated strand assimilation without background annealing

RAD51 is the central strand exchange recombinase in somatic homologous recombination, providing genomic stability and promoting resistance to DNA damage. An important tool for mechanistic studies of RAD51 is the D-loop or strand assimilation assay, which measures the ability of RAD51-coated single-stranded DNA (ssDNA) to search for, invade and exchange ssDNA strands with a homologous duplex DNA target. As cancer cells generally overexpress RAD51, the D-loop assay has also emerged as an important tool in oncologic drug design programs for targeting RAD51. Previous studies have adapted the traditional gel-based D-loop assay by using fluorescence-based substrates, which in principle allow for use in high-throughput screening platforms. However, these existing D-loop methods depend on linear oligonucleotide DNA duplex targets, and these substrates enable recombinase-independent ssDNA annealing that can obscure the recombinase-dependent strand assimilation signal. This compelled us to fundamentally re-design this assay, using a fluorescent target substrate that consists of a covalently closed linear double-hairpin dsDNA. This new microplate-based method represents a fast, inexpensive and non-radioactive alternative to existing D-loop assays. It provides accurate kinetic analysis of strand assimilation in high-throughput and performs well with human RAD51 and Escherichia coli RecA protein. This advance will aid in both mechanistic studies of homologous recombination and drug screening programs.     

Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule

The DNA strand-exchange reactions defining homologous recombination involve transient, nonuniform allosteric interactions between recombinase proteins and their DNA substrates. To study these mechanistic aspects of homologous recombination, we produced functional fluorescent human RAD51 recombinase and visualized recombinase interactions with single DNA molecules in both static and dynamic conditions. We observe that RAD51 nucleates filament formation at multiple sites on double-stranded DNA. This avid nucleation results in multiple RAD51 filament segments along a DNA molecule. Analysis of fluorescent filament patch size and filament kinks from scanning force microscopy (SFM) images indicate nucleation occurs minimally once every 500 bp. Filament segments did not rearrange along DNA, indicating tight association of the ATP-bound protein. The kinetics of filament disassembly was defined by activating ATP hydrolysis and following individual filaments in real time.