Single-molecule studies on the mechanical interplay between DNA supercoiling and H-NS DNA architectural properties

The Escherichia coli H-NS protein is a major nucleoid-associated protein that is involved in chromosomal DNA packaging and gene regulatory functions. These biological processes are intimately related to the DNA supercoiling state and thus suggest a direct relationship between H-NS binding and DNA supercoiling. Here, we show that H-NS, which has two distinct DNA-binding modes, is able to differentially regulate DNA supercoiling. H-NS DNA-stiffening mode caused by nucleoprotein filament formation is able to suppress DNA plectoneme formation during DNA supercoiling. In contrast, when H-NS is in its DNA-bridging mode, it is able to promote DNA plectoneme formation during DNA supercoiling. In addition, the DNA-bridging mode is able to block twists diffusion thus trapping DNA in supercoiled domains. Overall, this work reveals the mechanical interplay between H-NS and DNA supercoiling which provides insights to H-NS organization of chromosomal DNA based on its two distinct DNA architectural properties.     

Sequence-specific interactions of Rep proteins with ssDNA in the AT-rich region of the plasmid replication origin

The DNA unwinding element (DUE) is a sequence rich in adenine and thymine residues present within the origin region of both prokaryotic and eukaryotic replicons. Recently, it has been shown that this is the site where bacterial DnaA proteins, the chromosomal replication initiators, form a specific nucleoprotein filament. DnaA proteins contain a DNA binding domain (DBD) and belong to the family of origin binding proteins (OBPs). To date there has been no data on whether OBPs structurally different from DnaA can form nucleoprotein complexes within the DUE. In this work we demonstrate that plasmid Rep proteins, composed of two Winged Helix domains, distinct from the DBD, specifically bind to one of the strands of ssDNA within the DUE. We observed nucleoprotein complexes formed by these Rep proteins, involving both dsDNA containing the Rep-binding sites (iterons) and the strand-specific ssDNA of the DUE. Formation of these complexes required the presence of all repeated sequence elements located within the DUE. Any changes in these repeated sequences resulted in the disturbance in Rep-ssDNA DUE complex formation and the lack of origin replication activity in vivo or in vitro.     

Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function

Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination.     

Visualizing the Disassembly of S. cerevisiae Rad51 Nucleoprotein Filaments

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals. The assembly and disassembly of the Rad51 nucleoprotein filaments were visualized by tracking the location of the labeled DNA end in real time. Using this approach, we have analyzed yeast Rad51 under a variety of different reaction conditions to assess parameters that impact the stability of the nucleoprotein filament. We show that Rad51 readily dissociates from DNA in the presence of ADP or in the absence of nucleotide cofactor, but that free ATP in solution confers a fivefold increase in the stability of the nucleoprotein filaments. We also probe how protein dissociation is coupled to ATP binding and hydrolysis by examining the effects of ATP concentration, and by the use of the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido) triphosphate and ATPase active-site mutants. Finally, we demonstrate that the Rad51 gain-of-function mutant I345T dissociates from DNA with kinetics nearly identical to that of wild-type Rad51, but assembles 30% more rapidly. Together, these results provide a framework for studying the biochemical behaviors of S. cerevisiae Rad51 nucleoprotein filaments at the single-molecule level.     

The N-terminal domain of Escherichia coli RecA have multiple functions in promoting homologous recombination

Escherichia coli RecA mediates homologous recombination, a process essential to maintaining genome integrity. In the presence of ATP, RecA proteins bind a single-stranded DNA (ssDNA) to form a RecA-ssDNA presynaptic nucleoprotein filament that captures donor double-stranded DNA (dsDNA), searches for homology, and then catalyzes the strand exchange between ssDNA and dsDNA to produce a new heteroduplex DNA. Based upon a recently reported crystal structure of the RecA-ssDNA nucleoprotein filament, we carried out structural and functional studies of the N-terminal domain (NTD) of the RecA protein. The RecA NTD was thought to be required for monomer-monomer interaction. Here we report that it has two other distinct roles in promoting homologous recombination. It first facilitates the formation of a RecA-ssDNA presynaptic nucleoprotein filament by converting ATP to an ADP-Pi intermediate. Then, once the RecA-ssDNA presynaptic nucleoprotein filament is stably assembled in the presence of ATPγS, the NTD is required to capture donor dsDNA. Our results also suggest that the second function of NTD may be similar to that of Arg243 and Lys245, which were implicated earlier as binding sites of donor dsDNA. A two-step model is proposed to explain how a RecA-ssDNA presynaptic nucleoprotein filament interacts with donor dsDNA.     

The Single-Stranded DNA Binding Protein of Sulfolobus solfataricus Acts in the Presynaptic Step of Homologous Recombination

Homologous recombination is an important pathway in the repair of DNA double-strand breaks in all organisms. In mesophiles, single-stranded DNA binding proteins (SSBs) are believed to be involved in the removal of single-stranded DNA (ssDNA) secondary structure during the presynaptic step of homologous recombination, facilitating the formation of a contiguous Rad51/RecA nucleoprotein filament. Here we report a role for the thermophilic archaeal Sulfolobus solfataricus SSB (SsoSSB) in the presynaptic step of homologous recombination. We have identified multiple quaternary structural forms of this protein in vivo and examined the activity of SsoSSB with the strand-exchange protein S. solfataricus RadA (SsoRadA). Using gel-shift analysis, we found that the two major forms of SsoSSB have different DNA binding affinities and site sizes. Biochemical examination of the monomeric form of SsoSSB suggests that it has a minor role in presynapsis and may slightly inhibit the ssDNA-dependent ATPase activity of SsoRadA. The tetrameric form of SsoSSB, however, significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Order-of-addition experiments indicate that preincubation of tetrameric SsoSSB and SsoRadA prior to reaction initiation with ssDNA relieves the inhibition observed when SsoSSB is added either before or after SsoRadA. In addition, we demonstrate a direct interaction between SsoRadA and SsoSSB using coimmunoprecipitation. Taken together, these results suggest that a direct interaction between SsoSSB and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament.     

A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament

Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.     

Gene silencing H-NS paralogue StpA forms a rigid protein filament along DNA that blocks DNA accessibility

Nucleoid-associated proteins are bacterial proteins that are responsible for chromosomal DNA compaction and global gene regulation. One such protein is Escherichia coli Histone-like nucleoid structuring protein (H-NS) which functions as a global gene silencer. Whereas the DNA-binding mechanism of H-NS is well-characterized, its paralogue, StpA which is also able to silence genes is less understood. Here we show that StpA is similar to H-NS in that it is able to form a rigid filament along DNA. In contrast to H-NS, the StpA filament interacts with a naked DNA segment to cause DNA bridging which results in simultaneous stiffening and bridging of DNA. DNA accessibility is effectively blocked after the formation of StpA filament on DNA, suggesting rigid filament formation is the important step in promoting gene silencing. We also show that >1 mM magnesium promotes higher order DNA condensation, suggesting StpA may also play a role in chromosomal DNA packaging.     

Higher order oligomerization is required for H-NS family member MvaT to form gene-silencing nucleoprotein filament

MvaT from Pseudomonas aeruginosa is a member of the histone-like nucleoid structuring protein (H-NS) family of nucleoid-associated proteins widely spread among Gram-negative bacteria that functions to repress the expression of many genes. Recently, it was reported that H-NS from Escherichia coli can form rigid nucleoproteins filaments on DNA, which are important for their gene-silencing function. This raises a question whether the gene-silencing function of MvaT, which has only ∼18% sequence similarity to H-NS, is also based on the formation of nucleoprotein filaments. Here, using magnetic tweezers and atomic force microscopy imaging, we demonstrate that MvaT binds to DNA through cooperative polymerization to form a nucleoprotein filament that can further organize DNA into hairpins or higher-order compact structures. Furthermore, we studied DNA binding by MvaT mutants that fail to repress gene expression in P. aeruginosa because they are specifically defective for higher-order oligomer formation. We found that, although the mutants can organize DNA into compact structures, they fail to form rigid nucleoprotein filaments. Our findings suggest that higher-order oligomerization of MvaT is required for the formation of rigid nucleoprotein filaments that silence at least some target genes in P. aeruginosa. Further, our findings suggest that formation of nucleoprotein filaments provide a general structural basis for the gene-silencing H-NS family members.     

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament

Human RAD51 is a key protein in the repair of DNA by homologous recombination. Its assembly onto DNA, which induces changes in DNA structure, results in the formation of a nucleoprotein filament that forms the basis of strand exchange. Here, we determine the structural and mechanical properties of RAD51-dsDNA filaments. Our measurements use two recently developed magnetic tweezers assays, freely orbiting magnetic tweezers and magnetic torque tweezers, designed to measure the twist and torque of individual molecules. By directly monitoring changes in DNA twist on RAD51 binding, we determine the unwinding angle per RAD51 monomer to be 45°, in quantitative agreement with that of its bacterial homolog, RecA. Measurements of the torque that is built up when RAD51-dsDNA filaments are twisted show that under conditions that suppress ATP hydrolysis the torsional persistence length of the RAD51-dsDNA filament exceeds that of its RecA counterpart by a factor of three. Examination of the filament’s torsional stiffness for different combinations of divalent ions and nucleotide cofactors reveals that the Ca²⁺ ion, apart from suppressing ATPase activity, plays a key role in increasing the torsional stiffness of the filament. These quantitative measurements of RAD51-imposed DNA distortions and accumulated mechanical stress suggest a finely tuned interplay between chemical and mechanical interactions within the RAD51 nucleoprotein filament.     

CRISPR-dependent Base Editing Screens Identify Separation of Function Mutants of RADX with Altered RAD51 Regulatory Activity

RAD51 forms nucleoprotein filaments to promote homologous recombination, replication fork reversal, and fork protection. Numerous factors regulate the stability of these filaments and improper regulation leads to genomic instability and ultimately disease including cancer. RADX is a single stranded DNA binding protein that modulates RAD51 filament stability. Here, we utilize a CRISPR-dependent base editing screen to tile mutations across RADX to delineate motifs required for RADX function. We identified separation of function mutants of RADX that bind DNA and RAD51 but have a reduced ability to stimulate its ATP hydrolysis activity. Cells expressing these RADX mutants accumulate RAD51 on chromatin, exhibit replication defects, have reduced growth, accumulate DNA damage, and are hypersensitive to DNA damage and replication stress. These results indicate that RADX must promote RAD51 ATP turnover to regulate RAD51 and genome stability during DNA replication.     

Torque-limited RecA polymerization on dsDNA

The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA–DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 ± 0.7 k_bT. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.     

Characterization of a potent dominant negative mutant variant of Rad51 in Ustilago maydis

Rad51 serves to maintain and protect integrity of the genome through its actions in DNA repair and replication fork protection. The active form of Rad51 is a nucleoprotein filament consisting of chains of protomer units arranged linearly along single-stranded DNA. In a mutant screen using Ustilago maydis as an experimental system we identified a novel variant of Rad51, in which an amino acid change near the protomer–protomer interaction interface confers a strong trans dominant inhibitory effect on resistance to DNA damaging agents and proficiency in homologous recombination. Modeling studies of the mutated residue D161Y suggested that steric interference with surrounding residues was the likely cause of the inhibitory effect. Changes of two nearby residues, predicted from the modeling to minimize steric clashes, mitigated the inhibition of DNA repair. Direct testing of purified Rad51^D161Y protein in defined biochemical reactions revealed it to be devoid of DNA-binding activity itself, but capable of interfering with Rad51^WT in formation and maintenance of nucleoprotein filaments on single-stranded DNA and in DNA strand exchange. Rad51^D161Y protein appears to be unable to self-associate in solution and defective in forming complexes with the U. maydis BRCA2 ortholog.     

Interaction of single-stranded DNA with the second DNA-binding site of the RecA nucleoprotein filament

RecA first forms a filament on single-stranded DNA (ssDNA), thereby forming the first site for ssDNA binding and, simultaneously, the second site for binding double-stranded DNA (dsDNA). Then, the nucleoprotein filament interacts with dsDNA, although it can bind ssDNA as well. The resulting complex searches for homology sites and performs strand exchange between homologous DNA molecules. The interaction of various ssDNAs with the second DNA-recognizing site of RecA was studied by gradually increasing the structural complexity of the DNA ligand. Recognizing ssDNA with the second site, the protein interacts with each nucleotide of the ligand, forming contacts with both internucleotide phosphate groups and nitrogen bases. Pyrimidine oligonucleotides d(pC) _n and d(pT) _n interacted with the second site of the RecA filament more efficiently than d(pA) _n did. This was due to a more efficient interaction of the RecA filament with the 5′-terminal nucleotide of pyrimidinic DNA and to the difference in specific conformational changes of the nucleoprotein filament in the presence of purinic and pyrimidinic DNAs. A comparison of thermodynamic characteristics of DNA recognition at the first and second DNA-binding sites of the filament showed that, at n > 10, d(pC) _n and d(pN) _n were bound at the second site less tightly than at the first site. At n > 20, the second site bound d(pA) _n more efficiently than the first site. The difference in d(pN) _n affinity for the first and second sites increased monotonically with increasing n. Possible mechanisms of a RecA-dependent search for homology and DNA strand exchange are discussed.     

Single-Molecule Study on Histone-Like Nucleoid-Structuring Protein (H-NS) Paralogue in Pseudomonas aeruginosa: MvaU Bears DNA Organization Mode Similarities to MvaT

Pseudomonas aeruginosa contains two distinct members of H-NS family of nucleoid-structuring proteins: MvaT and MvaU. Together, these proteins bind to the same regions of the chromosome and function coordinately in the regulation of hundreds of genes. Due to their structural similarity, they can associate to form heteromeric complexes. These findings left us wondering whether they bear similar DNA binding properties that underlie their gene-silencing functions. Using single-molecule stretching and imaging experiments, we found striking similarities in the DNA organization modes of MvaU compared to the previously studied MvaT. MvaU can form protective nucleoprotein filaments that are insensitive to environmental factors, consistent with its role as a repressor of gene expression. Similar to MvaT, MvaU filament can mediate DNA bridging while excessive MvaU can cause DNA aggregation. The almost identical DNA organization modes of MvaU and MvaT explain their functional redundancy, and raise an interesting question regarding the evolutionary benefits of having multiple H-NS paralogues in the Pseudomonas genus.     

An archaeal RadA paralog influences presynaptic filament formation

Recombinases of the RecA family play vital roles in homologous recombination, a high-fidelity mechanism to repair DNA double-stranded breaks. These proteins catalyze strand invasion and exchange after forming dynamic nucleoprotein filaments on ssDNA. Increasing evidence suggests that stabilization of these dynamic filaments is a highly conserved function across diverse species. Here, we analyze the presynaptic filament formation and DNA binding characteristics of the Sulfolobus solfataricus recombinase SsoRadA in conjunction with the SsoRadA paralog SsoRal1. In addition to constraining SsoRadA ssDNA-dependent ATPase activity, the paralog also enhances SsoRadA ssDNA binding, effectively influencing activities necessary for presynaptic filament formation. These activities result in enhanced SsoRadA-mediated strand invasion in the presence of SsoRal1 and suggest a filament stabilization function for the SsoRal1 protein.     

A chimeric Rec-A protein that implicates non-Watson-Crick interactions in homologous pairing.

The helical filament formed by RecA protein on single-stranded DNA plays an important role in homologous recombination and pairs with a complementary single strand or homologous duplex DNA. The RecA nucleoprotein filament also recognizes an identical single strand. The chimeric protein, RecAc38, forms a nucleoprotein filament that recognizes a complementary strand but is defective in recognition of duplex DNA, and is associated with phenotypic defects in repair and recombination. As described here, RecAc38 nucleoprotein filament is also defective in recognition of an identical strand, either when the filament has within it a single strand or duplex DNA. A model that postulates three DNA binding sites rationalizes these observations and suggests that the third binding site mediates non-Watson-Crick interactions that are instrumental in recognition of homology in duplex DNA.     

Stability of blocked replication forks in vivo

Replication of chromosomal DNA must be carried out to completion in order for a cell to proliferate. However, replication forks can stall during this process for a variety of reasons, including nucleoprotein ‘roadblocks’ and DNA lesions. In these circumstances the replisome copying the DNA may disengage from the chromosome to allow various repair processes to restore DNA integrity and enable replication to continue. Here, we report the in vivo stability of the replication fork when it encounters a nucleoprotein blockage in Escherichia coli. Using a site-specific and reversible protein block system in conjunction with the temperature sensitive DnaC helicase loader and DnaB replicative helicase, we monitored the disappearance of the Y-shaped DNA replication fork structures using neutral-neutral 2D agarose gels. We show the replication fork collapses within 5 min of encountering the roadblock. Therefore, the stalled replication fork does not pause at a block in a stable confirmation for an extended period of time as previously postulated.