Nucleoprotein Filament Formation Research Articles

BRCA2 stabilises RAD51 and DMC1 nucleoprotein filaments through a conserved interaction mode

BRCA2 is essential for DNA repair by homologous recombination in mitosis and meiosis. It interacts with recombinases RAD51 and DMC1 to facilitate the formation of nucleoprotein filaments on resected DNA ends that catalyse recombination-mediated repair. BRCA2’s BRC repeats bind and disrupt RAD51 and DMC1 filaments, whereas its PhePP motifs bind recombinases and stabilise their nucleoprotein filaments. However, the mechanism of filament stabilisation has hitherto remained unknown. Here, we report the crystal structure of a BRCA2-DMC1 complex, revealing how core interaction sites of PhePP motifs bind to recombinases. The interaction mode is conserved for RAD51 and DMC1, which selectively bind to BRCA2’s two distinct PhePP motifs via subtly divergent binding pockets. PhePP motif sequences surrounding their core interaction sites protect nucleoprotein filaments from BRC-mediated disruption. Hence, we report the structural basis of how BRCA2’s PhePP motifs stabilise RAD51 and DMC1 nucleoprotein filaments for their essential roles in mitotic and meiotic recombination.

Mei5-Sae3 stabilizes Dmc1 nucleating clusters for efficient Dmc1 assembly on RPA-coated single-stranded DNA.

Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5-Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5-Sae3 are interdependent in their colocalization on the chromosomes. However, the mechanistic role of Mei5-Sae3 in mediating Dmc1 activity remains unclear. We used single-molecule fluorescence resonance energy transfer and colocalization single-molecule spectroscopy experiments to elucidate how Mei5-Sae3 stimulates Dmc1 assembly on ssDNA and RPA-coated ssDNA. We showed that Mei5-Sae3 stabilized Dmc1 nucleating clusters with two to threemolecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5-Sae3 also stimulated Dmc1 assembly on RPA-coated DNA. Using green fluorescent protein-labeled RPA, we showed the coexistence of an intermediate with Dmc1 and RPA on ssDNA before RPA dissociation. Moreover, the displacement efficiency of RPA depended on Dmc1 concentration, and its dependence was positively correlated with the stability of Dmc1 clusters on short ssDNA. These findings suggest a molecular model that Mei5-Sae3 mediates Dmc1 binding on RPA-coated ssDNA by stabilizing Dmc1 nucleating clusters, thus altering RPA dynamics on DNA to promote RPA dissociation.

Structure of RADX and mechanism for regulation of RAD51 nucleofilaments

Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed single strand DNA (ssDNA). To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX (RPA-related RAD51-antagonist on the X chromosome) is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here, we present a structure-based investigation of RADX's mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration-dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (cryo-EM) from maps in the 2 to 4 Å range. The structure reveals the molecular basis for RADX oligomerization and the coupled multi-valent binding of ssDNA binding. The interaction of RADX with RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the end of RAD51 filaments.

In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase.

Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation.

The protein phosphatase EYA4 promotes homologous recombination (HR) through dephosphorylation of tyrosine 315 on RAD51.

Efficient DNA repair and limitation of genome rearrangements rely on crosstalk between different DNA double-strand break (DSB) repair pathways, and their synchronization with the cell cycle. The selection, timingand efficacy of DSB repair pathways are influenced by post-translational modifications of histones and DNA damage repair (DDR) proteins, such as phosphorylation. While the importance of kinases and serine/threonine phosphatases in DDR have been extensively studied, the role of tyrosine phosphatases in DNA repair remains poorly understood. In this study, we have identified EYA4 as the protein phosphatase that dephosphorylates RAD51 on residue Tyr315. Through its Tyr phosphatase activity, EYA4 regulates RAD51 localization, presynaptic filament formation, foci formation, and activity. Thus, it is essential for homologous recombination (HR) at DSBs. DNA binding stimulates EYA4 phosphatase activity. Depletion of EYA4 decreases single-stranded DNA accumulation following DNA damage and impairs HR, while overexpression of EYA4 in cells promotes dephosphorylation and stabilization of RAD51, and thereby nucleoprotein filament formation. Our data have implications for a pathological version of RAD51 in EYA4-overexpressing cancers.

Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes

Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.

Modeling the Homologous Recombination Process: Methods, Successes and Challenges.

Homologous recombination (HR) is a fundamental process common to all species. HR aims to faithfully repair DNA double strand breaks. HR involves the formation of nucleoprotein filaments on DNA single strands (ssDNA) resected from the break. The nucleoprotein filaments search for homologous regions in the genome and promote strand exchange with the ssDNA homologous region in an unbroken copy of the genome. HR has been the object of intensive studies for decades. Because multi-scale dynamics is a fundamental aspect of this process, studying HR is highly challenging, both experimentally and using computational approaches. Nevertheless, knowledge has built up over the years and has recently progressed at an accelerated pace, borne by increasingly focused investigations using new techniques such as single molecule approaches. Linking this knowledge to the atomic structure of the nucleoprotein filament systems and the succession of unstable, transient intermediate steps that takes place during the HR process remains a challenge; modeling retains a very strong role in bridging the gap between structures that are stable enough to be observed and in exploring transition paths between these structures. However, working on ever-changing long filament systems submitted to kinetic processes is full of pitfalls. This review presents the modeling tools that are used in such studies, their possibilities and limitations, and reviews the advances in the knowledge of the HR process that have been obtained through modeling. Notably, we will emphasize how cooperative behavior in the HR nucleoprotein filament enables modeling to produce reliable information.

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.

A real-time fluorescent gp32 probe-based assay for monitoring single-stranded DNA-dependent DNA processing enzymes

Single-stranded DNA (ssDNA) generated during DNA replication, recombination and damage repair reactions is an important intermediate and ssDNA-binding proteins that binds these intermediates coordinate various DNA metabolic processes. Mechanistic details of these ssDNA-dependent processes can be explored by monitoring the generation and consumption of ssDNA in real time. In this work, a fluorescein-labeled gp32-based sensor was employed to continuously monitor various aspects of ssDNA-dependent DNA replication and recombination processes in real time. The gp32 protein probe displayed high sensitivity and specificity to a variety of ssDNA-dependent processes of T4 phage. Several applications of the probe are illustrated here: the solution dynamics of ssDNA-binding protein, protein-protein and protein-DNA interactions involving gp32 protein and its mode of interaction, ssDNA translocation and protein displacement activities of helicases, primer extension activity of DNA polymerase holoenzyme and nucleoprotein filament formation during DNA recombination. The assay has identified new protein-protein interactions of gp32 during T4 replication and recombination. The fluorescent probe described here can thus be used as a universal probe for monitoring in real time various ssDNA-dependent processes, which is based on a well-characterized and easy-to-express bacteriophage T4 gene 32 protein, gp32.

The Inability to Disassemble Rad51 Nucleoprotein Filaments Leads to Aberrant Mitosis and Cell Death.

The proper maintenance of genetic material is essential for the survival of living organisms. One of the main safeguards of genome stability is homologous recombination involved in the faithful repair of DNA double-strand breaks, the restoration of collapsed replication forks, and the bypass of replication barriers. Homologous recombination relies on the formation of Rad51 nucleoprotein filaments which are responsible for the homology-based interactions between DNA strands. Here, we demonstrate that without the regulation of these filaments by Srs2 and Rad54, which are known to remove Rad51 from single-stranded and double-stranded DNA, respectively, the filaments strongly inhibit damage-associated DNA synthesis during DNA repair. Furthermore, this regulation is essential for cell survival under normal growth conditions, as in the srs2Δ rad54Δ mutants, unregulated Rad51 nucleoprotein filaments cause activation of the DNA damage checkpoint, formation of mitotic bridges, and loss of genetic material. These genome instability features may stem from the problems at stalled replication forks as the lack of Srs2 and Rad54 in the presence of Rad51 nucleoprotein filaments impedes cell recovery from replication stress. This study demonstrates that the timely and efficient disassembly of recombination machinery is essential for genome maintenance and cell survival.

A metal ion-dependent mechanism of RAD51 nucleoprotein filament disassembly

The RAD51 ATPase polymerizes on single-stranded DNA to form nucleoprotein filaments (NPFs) that are critical intermediates in the reaction of homologous recombination. ATP binding maintains the NPF in a competent conformation for strand pairing and exchange. Once strand exchange is completed, ATP hydrolysis licenses the filament for disassembly. Here we show that the ATP-binding site of the RAD51 NPF contains a second metal ion. In the presence of ATP, the metal ion promotes the local folding of RAD51 into the conformation required for DNA binding. The metal ion is absent in the ADP-bound RAD51 filament, that rearranges in a conformation incompatible with DNA binding. The presence of the second metal ion explains how RAD51 couples the nucleotide state of the filament to DNA binding. We propose that loss of the second metal ion upon ATP hydrolysis drives RAD51 dissociation from the DNA and weakens filament stability, contributing to NPF disassembly.

Divalent metal cofactors differentially modulate RadA-mediated strand invasion and exchange in Saccharolobus solfataricus.

Central to the universal process of recombination, RecA family proteins form nucleoprotein filaments to catalyze production of heteroduplex DNA between substrate ssDNAs and template dsDNAs. ATP binding assists the filament in assuming the necessary conformation for forming heteroduplex DNA, but hydrolysis is not required. ATP hydrolysis has two identified roles which are not universally conserved: promotion of filament dissociation and enhancing flexibility of the filament. In this work, we examine ATP utilization of the RecA family recombinase SsoRadA from Saccharolobus solfataricus to determine its function in recombinase-mediated heteroduplex DNA formation. Wild-type SsoRadA protein and two ATPase mutant proteins were evaluated for the effects of three divalent metal cofactors. We found that unlike other archaeal RadA proteins, SsoRadA-mediated strand exchange is not enhanced by Ca2+. Instead, the S. solfataricus recombinase can utilize Mn2+ to stimulate strand invasion and reduce ADP-binding stability. Additionally, reduction of SsoRadA ATPase activity by Walker Box mutation or cofactor alteration resulted in a loss of large, complete strand exchange products. Depletion of ADP was found to improve initial strand invasion but also led to a similar loss of large strand exchange events. Our results indicate that overall, SsoRadA is distinct in its use of divalent cofactors but its activity with Mn2+ shows similarity to human RAD51 protein with Ca2+.

Rtt105 regulates RPA function by configurationally stapling the flexible domains

Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

Repair of DNA double-strand breaks in plant meiosis: role of eukaryotic RecA recombinases and their modulators.

Homologous recombination during meiosis is crucial for the DNA double-strand breaks (DSBs) repair that promotes the balanced segregation of homologous chromosomes and enhances genetic variation. In most eukaryotes, two recombinases RAD51 and DMC1 form nucleoprotein filaments on single-stranded DNA generated at DSB sites and play a central role in the meiotic DSB repair and genome stability. These nucleoprotein filaments perform homology search and DNA strand exchange to initiate repair using homologous template-directed sequences located elsewhere in the genome. Multiple factors can regulate the assembly, stability, and disassembly of RAD51 and DMC1 nucleoprotein filaments. In this review, we summarize the current understanding of the meiotic functions of RAD51 and DMC1 and the role of their positive and negative modulators. We discuss the current models and regulators of homology searches and strand exchange conserved during plant meiosis. Manipulation of these repair factors during plant meiosis also holds a great potential to accelerate plant breeding for crop improvements and productivity.

Bacterial H-NS contacts DNA at the same irregularly spaced sites in both bridged and hemi-sequestered linear filaments

SummaryGene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD–DNA contacts in linear and bridged filaments are uncertain. To characterize the H-NS DBD contacts that silence transcription by RNA polymerase, we combined ·OH footprinting, molecular dynamics, statistical modeling, and DBD mapping using a chemical nuclease (Fe2+-EDTA) tethered to the DBDs (TEN-map). We find that H-NS DBDs contact DNA at indistinguishable locations in bridged or linear filaments and that the DBDs vary in orientation and position with ∼10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged H-NS switching. Linear filaments able to inhibit only transcription initiation switch to bridged filaments able to inhibit both initiation and elongation using the same irregularly spaced DNA contacts.

Single-molecule characterization of compressed RecA nucleoprotein filaments

RecA is a central enzyme of homologous recombination in bacteria, which plays a major role in DNA repair, natural transformation and SOS-response activation. RecA forms nucleoprotein filaments on single-stranded DNA with a highly conserved architecture that is also shared by eukaryotic recombinases. One of the key features of these filaments is the ability to switch between stretched and compressed conformations in response to ATP binding and hydrolysis. However, the functional role of such conformational changes is not fully understood. Structural data revealed that in the absence of ATP RecA binds DNA with the stoichiometry of 5 nucleotides per one monomer, while in the presence of ATP the binding stoichiometry is 3:1. Such differences suggest incompatibility of the active and inactive conformations, yet dynamic single-molecule studies demonstrated that ATP and apo conformations can be directly interconvertible. In the present work we use a single-molecule approach to address the features of inactive RecA nucleoprotein filaments formed de novo in the absence of nucleotide cofactors. We show that compressed RecA-DNA filaments can exist with both 5:1 and 3:1 binding stoichiometry which is determined by conditions of the filament assembly. However, only a 3:1 stoichiometry allows direct interconvertibility with the active ATP-bound conformation.

Graphene oxide nanosheet-mediated fluorescent RPA “turn-on” biosensor for rapid RNAi transgenic plant detection

In this paper preliminarily verified that graphene oxide (GO) nanomaterials enhanced the recombinase polymerase amplification (RPA). GO nanosheets improved the efficiency of RPA amplification by absorbing ingredients to induce local aggregation. The recombinase initially aggregated with the primers to form nucleoprotein filaments, absorbed on the GO nanosheets, changing the structure. Therefore, an isothermal fluorescence biosensor was developed based on GO nanosheets enhanced the RPA to detect RNA interference (RNAi) transgenic plants. FAM-labeled primers were absorbed and quenched by the GO nanosheets. After amplification, the primers were extended into double-stranded DNA, detaching from the GO surface to recover the fluorescent signal. The biosensor displayed high sensitivity and selectivity and showed an excellent relationship ranging from 1.5 to 100 ng of genome DNA, with a detection limit (LOD) of 1.5 ng. Consequently, the biosensor provides an enhanced isothermal method for detecting genetically modified (GM) products and exhibits significant potential for molecular detection.

A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.

Dual PARP and RAD51 Inhibitory Drug Conjugates Show Synergistic and Selective Effects on Breast Cancer Cells.

The genetic principle of synthetic lethality has most successfully been exploited in therapies engaging Poly-ADP-ribose-polymerase (PARP) inhibitors to treat patients with homologous recombination (HR)-defective tumors. In this work, we went a step further following the idea of a local molecular cooperation and designed hybrid compounds M1–M3. The drug conjugates M1–M3 combine Olaparib, the first PARP inhibitor approved for clinical use, with Cpd 1, an inhibitor of RAD51 that blocks its HR functions and yet permits RAD51 nucleoprotein filament formation on single-stranded DNA. While in M2 and M3, the parental drugs are linked by -CO-(CH2)n-CO-spacers (n = 2 and 4, respectively), they are directly merged omitting the piperazine ring of Olaparib in M1. Monitoring anti-survival effects of M1–M3 in six breast cancer cell lines of different molecular subtypes showed that in each cell line, at least one of the drug conjugates decreased viability by one to two orders of magnitude compared with parental drugs. While triple-negative breast cancer (TNBC) cells with frequent BRCA1 pathway dysfunction were sensitive to spacer-linked hybrid compounds M1 and M2 regardless of their HR capacities, non-TNBC cells were responsive to the merged drug conjugate M1 only, suggesting different spatial requirements for dual inhibition in these two groups of cell lines. These results demonstrate that, depending on chemical linkage, dual PARP1-RAD51 inhibitory drugs can either sensitize non-TNBC and re-sensitize TNBC cells, or discriminate between these groups of cells.

The regulation mechanism of the C-terminus of RecA proteins during DNA strand-exchange process

The C-terminus of Escherichia coli RecA protein can affect the DNA binding affinity, interact with accessory proteins, and regulate the RecA activity. A substantial upward shift in the pH-reaction profile of RecA-mediated DNA strand-exchange reactions was observed for C-terminal-truncated E. coli ΔC17 RecA, Deinococcus radiodurans RecA, and Deinococcus ficus RecA. Here, the process of RecA-mediated strand exchange from the beginning to the end was investigated with florescence resonance energy transfer and tethered particle motion experiments to determine the detailed regulation mechanism. RecA proteins with a shorter C-terminus possess more stable nuclei, higher DNA binding affinities, and lower protonation requirements for the formation of nucleoprotein filaments. Moreover, more stable synaptic complexes in the homologous sequence searching process were also observed for RecA proteins with a shorter C-terminus. Our results suggest that the C-terminus of RecA proteins regulates not only the formation of RecA nucleoprotein filaments but also the entrance of secondary DNA into RecA nucleoprotein filaments.