ssDNA-binding Domain Research Articles

Molecular basis and functional consequences of the interaction between the base excision repair DNA glycosylase NEIL1 and RPA

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd ∼ 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against a thymine glycol lesion in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.

The intrinsically disordered linker in the single-stranded DNA-binding protein influences DNA replication restart and recombination pathways in Escherichia coli K-12.

Tetrameric single-stranded (ss) DNA-binding proteins (SSBs) stabilize ssDNA intermediates formed during genome maintenance reactions in Bacteria. SSBs also recruit proteins important for these processes through direct SSB-protein interactions, including proteins involved in DNA replication restart and recombination processes. SSBs are composed of an N-terminal oligomerization and ssDNA-binding domain, a C-terminal acidic tip that mediates SSB-protein interactions, and an internal intrinsically disordered linker (IDL). Deletions and insertions into the IDL are well tolerated with few phenotypes, although the largest deletions and insertions exhibit some sensitivity to DNA-damaging agents. To define specific DNA metabolism processes dependent on IDL length, ssb mutants that lack 16, 26, 37, or 47 residues of the 57-residue IDL were tested for synthetic phenotypes with mutations in DNA replication restart or recombination genes. We also tested the impact of integrating a fluorescent domain within the SSB IDL using an ssb::mTur2 insertion mutation. Only the largest deletion tested or the insertion mutation causes sensitivity in any of the pathways. Mutations in two replication restart pathways (PriA-B1 and PriA-C) showed synthetic lethalities or small colony phenotypes with the largest deletion or insertion mutations. Recombination gene mutations del(recBCD) and del(ruvABC) show synthetic phenotypes only when combined with the largest ssb deletion. These results suggest that a minimum IDL length is important in some genome maintenance reactions in Escherichia coli. These include pathways involving PriA-PriB1, PriA-PriC, RecFOR, and RecG. The mTur2 insertion in the IDL may also affect SSB interactions in some processes, particularly the PriA-PriB1 and PriA-PriC replication restart pathways.IMPORTANCEssb is essential in Escherichia coli due to its roles in protecting ssDNA and coordinating genome maintenance events. While the DNA-binding core and acidic tip have well-characterized functions, the purpose of the intrinsically disordered linker (IDL) is poorly understood. In vitro studies have revealed that the IDL is important for cooperative ssDNA binding and phase separation. However, single-stranded (ss) DNA-binding protein (SSB) variants with large deletions and insertions in the IDL support normal cell growth. We find that the PriA-PriB1 and PriA-C replication restart, as well as the RecFOR- and RecG-dependent recombination, pathways are sensitive to IDL length. This suggests that cooperativity, phase separation, or a longer spacer between the core and acidic tip of SSB may be important for specific cellular functions.

The Role of S. cerevisiae Sub1/PC4 in Transcription Elongation Depends on the C-Terminal Region and Is Independent of the ssDNA Binding Domain.

Saccharomyces cerevisiae Sub1 (ScSub1) has been defined as a transcriptional stimulatory protein due to its homology to the ssDNA binding domain (ssDBD) of human PC4 (hPC4). Recently, PC4/Sub1 orthologues have been elucidated in eukaryotes, prokaryotes, and bacteriophages with functions related to DNA metabolism. Additionally, ScSub1 contains a unique carboxyl–terminal region (CT) of unknown function up to date. Specifically, it has been shown that Sub1 is required for transcription activation, as well as other processes, throughout the transcription cycle. Despite the progress that has been made in understanding the mechanism underlying Sub1′s functions, some questions remain unanswered. As a case in point: whether Sub1’s roles in initiation and elongation are differentially predicated on distinct regions of the protein or how Sub1′s functions are regulated. Here, we uncover some residues that are key for DNA–ScSub1 interaction in vivo, localized in the ssDBD, and required for Sub1 recruitment to promoters. Furthermore, using an array of genetic and molecular techniques, we demonstrate that the CT region is required for transcription elongation by RNA polymerase II (RNAPII). Altogether, our data indicate that Sub1 plays a dual role during transcription—in initiation through the ssDBD and in elongation through the CT region.

Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity.

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed “jumping” behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes.

Single-Stranded DNA-Binding Proteins in the Archaea.

Single-stranded (ss) DNA-binding proteins are found in all three domains of life where they play vital roles in nearly all aspects of DNA metabolism by binding to and stabilizing exposed ssDNA and acting as platforms onto which DNA-processing activities can assemble. The ssDNA-binding factors SSB and RPA are extremely well conserved across bacteria and eukaryotes, respectively, and comprise one or more OB-fold ssDNA-binding domains. In the third domain of life, the archaea, multiple types of ssDNA-binding protein are found with a variety of domain architectures and subunit compositions, with OB-fold ssDNA-binding domains being a characteristic of most, but not all. This chapter summarizes current knowledge of the distribution, structure, and biological function of the archaeal ssDNA-binding factors, highlighting key features shared between clades and those that distinguish the proteins of different clades from one another. The likely cellular functions of the proteins are discussed and gaps in current knowledge identified.

Phase separation by ssDNA binding protein controlled via protein−protein and protein−DNA interactions

Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL), and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the Escherichia coli SSB protein forms liquid-liquid phase-separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA, and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage.

Mechanistic insights from structure of Mycobacterium smegmatis topoisomerase I with ssDNA bound to both N- and C-terminal domains.

Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

E. coli Single-Stranded DNA Binding (SSB) Protein Undergoes Dynamic Liquid-Liquid Phase Separation Controlled via Protein-Protein and Protein-DNA Interactions

A number of eukaryotic proteins have recently been shown to form phase-separated liquid condensates (membrane-less organelles) playing key roles in normal cell physiology and stress tolerance as bioreactors, biomolecular filters, stress sensors or molecular reservoirs. However, it is still debated whether liquid-liquid phase separation (LLPS) is a fundamental process in bacteria as it is in eukaryotes. Here we report the striking observation that the E. coli SSB protein, a ubiquitous central player of practically all DNA metabolic processes, forms phase-separated condensates under physiological conditions. LLPS is generally driven by multivalent weak protein-protein or protein-nucleic acid interactions, mediated mostly by intrinsically disordered protein regions. We found that LLPS by the homotetrameric SSB protein is mediated via multifaceted inter-tetramer interactions involving all protein regions including the conserved ssDNA-binding domain, the intrinsically disordered linker (IDL) and the C-terminal peptide (CTP, a highly conserved protein-protein interaction motif binding to a variety of DNA metabolic proteins). SSB, ssDNA and SSB-interacting partners are highly concentrated within the phase-separated droplets, whereas LLPS is overall regulated by the stoichiometry of SSB and ssDNA. Our bioinformatics analysis indicates that the LLPS-forming propensity of the SSB IDL is broadly conserved across all major phylogenetic groups of Eubacteria. Together with recently observed dynamic spot-like subcellular localization patterns of SSB, our results suggest that bacterial cells store an abundant pool of SSB and SSB-interacting proteins in phase-separated condensates via a conserved mechanism. The discovered features enable rapid mobilization of SSB to ssDNA regions exposed upon DNA damage or metabolic processes to serve efficient repair, replication and recombination.

A key interaction with RPA orients XPA in NER complexes.

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD–RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

The Many Roles of Binding Cooperativity in the Control of DNA Replication

The Many Roles of Binding Cooperativity in the Control of DNA Replication

Protection of telomeres 1 proteins POT1a and POT1b can repress ATR signaling by RPA exclusion, but binding to CST limits ATR repression by POT1b

Comprised of telomeric TTAGGG repeats and shelterin, telomeres ensure that the natural ends of chromosomes remain impervious to the DNA damage response. Telomeres carry a long constitutive 3' overhang that can bind replication protein A (RPA) and activate the ATR Ser/Thr kinase (ATR), which induces cell cycle arrest. A single-stranded (ss) TTAGGG repeat-binding protein in mouse shelterin, POT1a, has been proposed to repress ATR signaling by preventing RPA binding. Repression of ATR at telomeres requires tethering of POT1a to the other shelterin subunits situated on the double-stranded (ds) telomeric DNA. The simplest model of ATR repression, the "tethered exclusion model," suggests that the only critical features of POT1a are its connection to shelterin and its binding to ss telomeric DNA. In agreement with the model, we show here that a shelterin-tethered variant of RPA70 (lacking the ATR recruitment domain) can repress ATR signaling at telomeres that lack POT1a. However, arguing against the tethered exclusion model, the nearly identical POT1b subunit of shelterin has been shown to be much less proficient than POT1a in repression of ATR. We now show that POT1b has the intrinsic ability to fully repress ATR but is prevented from doing so when bound to Ctc1, Stn1, Ten1 (CST), the complex needed for telomere end processing. These results establish that shelterin represses ATR with a tethered ssDNA-binding domain that excludes RPA from the 3' overhang and also reveal an unexpected effect of CST on the ability of POT1b to repress ATR.

Abstract B39: Small Molecule Disruption of RAD52 Rings as a Mechanism for Precision Medicine in BRCA Deficient Cancers

Abstract Suppression of RAD52 causes synthetic lethality in BRCA deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA deficient cancers. Citation Format: Richard T. Pomerantz. Small Molecule Disruption of RAD52 Rings as a Mechanism for Precision Medicine in BRCA Deficient Cancers [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B39.

Identification of a Small Molecule Inhibitor of RAD52 to Induce Synthetic Lethality in BRCA-Deficient Leukemias

Altered DNA repair mechanisms are responsible for survival of leukemia stem cells (LSCs) and/or leukemia progenitor cells (LPCs) accumulating numerous lethal DNA double-strand breaks (DSBs). DSBs resulting from stalled/broken replication forks in proliferating cells are primarily repaired by RAD51-mediated homologous recombination repair (HR), which depends on BRCA1-PALB2-BRCA2-RAD51 paralogs (BRCA pathway), while RAD52 pathway serves as redundant back-up mechanism. Enhanced self-renewal of LSCs and high proliferation rate of LPCs commit them to HR.It has been reported that inhibition of RAD52 either by the knockout, specific shRNA, or a small peptide aptamer induced synthetic lethality in BRCA pathway-deficient tumor cell lines and primary leukemia cells. Yet pharmacological inhibition of RAD52, which binds single-stranded DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated.Here, we applied high-throughput screening and structure-based selection followed by biochemical assays and computer modeling to identify three leading compounds: (1) 20264, (2) RU-0084339, and (3) D-I03. Compound 20264 appeared to interact with the hotspot in RAD52 DNA binding domain 1 to interfere with ssDNA binding. RU-0084339 is a major allosteric inhibitor of RAD52 ssDNA binding domain which disassembles undecamer ring structure of RAD52. D-I03 abrogated RAD52-mediated ssDNA annealing and ssDNA pairing. RAD52 small molecule inhibitor (RAD52smi) reduced recruitment of RAD52 to DNA damage-induced nuclear foci and suppressed RAD52-mediated DNA double-strand break (DSB) repair activity in cells with negligible effects on other DSB repair pathways. Importantly, RAD52smi selectively eliminated cancer cell lines carrying BRCA1/2 inactivating mutations.Since inactivating mutations in BRCA pathway are rare in leukemias, individual BRCA pathway-deficient leukemias were identified by Gene Expression and Mutation Analysis (GEMA). Gene Expression approach applied microarrays, qRT-PCR, and/or flow cytometry to identify individual leukemias displaying downregulation of at least one gene in BRCA pathway. On the other hand, Gene Mutation strategy detected individual leukemias expressing an oncogene causing downregulation of BRCA pathway gene(s) (e.g., BCR-ABL1, MLL-AF9, AML1-ETO - mediated downregulation of BRCA1 and/or BRCA2) and harboring inactivating mutations in BRCA pathway (e.g., BRCA2 = FANCD1, and other FA genes). BRCA-deficient cells from individual patients indentified by GEMA were selectively sensitive to RAD52smi alone or in combination with already approved cytotoxic drugs.RAD52 is a promising new target because it is "druggable" by small molecule inhibitors. Moreover, inhibition of RAD52 by genetic knockout and small peptide aptamer did not exert any major negative effects in normal cells and tissues. Altogether, this work provided foundation for precision medicine guided synthetic lethality in BRCA-deficient leukemias exerted by small molecule inhibitors targeting novel mechanism - RAD52 dependent DSB repair. DisclosuresNo relevant conflicts of interest to declare.

Small-Molecule Disruption of RAD52 Rings as a Mechanism for Precision Medicine in BRCA-Deficient Cancers

Suppression of RAD52 causes synthetic lethality in BRCA-deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-Dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA-deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small-molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA-deficient cancers.

Functional Dynamics in Replication Protein A DNA Binding and Protein Recruitment Domains

Replication Protein A (RPA) is an essential scaffold for many DNA processing machines; its function relies on its modular architecture. Here, we report (15)N-nuclear magnetic resonance heteronuclear relaxation analysis to characterize the movements of single-stranded (ss) DNA binding and protein interaction modules in the RPA70 subunit. Our results provide direct evidence for coordination of the motion of the tandem RPA70AB ssDNA binding domains. Moreover, binding of ssDNA substrate is found to cause dramatic reorientation and full coupling of inter-domain motion. In contrast, the RPA70N protein interaction domain remains structurally and dynamically independent of RPA70AB regardless of binding of ssDNA. This autonomy of motion between the 70N and 70AB modules supports a model in which the two binding functions of RPA are mediated fully independently, but remain differentially coordinated depending on the length of their flexible tethers. A critical role for linkers between the globular domains in determining the functional dynamics of RPA is proposed.

Direct single-molecule observations of local denaturation of a DNA double helix under a negative supercoil state.

Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA.

Intrinsically Disordered C-Terminal Tails of E. coli Single-Stranded DNA Binding Protein Regulate Cooperative Binding to Single-Stranded DNA

The homotetrameric Escherichia coli single-stranded DNA binding protein (SSB) plays a central role in DNA replication, repair and recombination. E. coli SSB can bind to long single-stranded DNA (ssDNA) in multiple binding modes using all four subunits [(SSB)65 mode] or only two subunits [(SSB)35 binding mode], with the binding mode preference regulated by salt concentration and SSB binding density. These binding modes display very different ssDNA binding properties with the (SSB)35 mode displaying highly cooperative binding to ssDNA. SSB tetramers also bind an array of partner proteins, recruiting them to their sites of action. This is achieved through interactions with the last 9 amino acids (acidic tip) of the intrinsically disordered linkers (IDLs) within the four C-terminal tails connected to the ssDNA binding domains. Here, we show that the amino acid composition and length of the IDL affects the ssDNA binding mode preferences of SSB protein. Surprisingly, the number of IDLs and the lengths of individual IDLs together with the acidic tip contribute to highly cooperative binding in the (SSB)35 binding mode. Hydrodynamic studies and atomistic simulations suggest that the E. coli SSB IDLs show a preference for forming an ensemble of globular conformations, whereas the IDL from Plasmodium falciparum SSB forms an ensemble of more extended random coils. The more globular conformations correlate with cooperative binding.

Bound or Free: Interaction of the C-Terminal Domain of Escherichia coli Single-Stranded DNA-Binding Protein (SSB) with the Tetrameric Core of SSB

Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A

Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (−flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (−flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (−flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (−flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3′-5′exo–) was promoted by DNA stretching with buffer flow.

Rad54 Functions as a Heteroduplex DNA Pump Modulated by Its DNA Substrates and Rad51 during D Loop Formation

The displacement loop (D loop) is the product of homology search and DNA strand invasion, constituting a central intermediate in homologous recombination (HR). In eukaryotes, the Rad51 DNA strand exchange protein is assisted in D loop formation by the Rad54 motor protein. Curiously, Rad54 also disrupts D loops. How these opposing activities are coordinated toward productive recombination is unknown. Moreover, a seemingly disparate function of Rad54 is removal of Rad51 from heteroduplex DNA (hDNA) to allow HR-associated DNA synthesis. Here, we uncover features of D loop formation/dissociation dynamics, employing Rad51 filaments formed on ssDNAs that mimic the physiological length and structure of in vivo substrates. The Rad54 motor is activated by Rad51 bound to synapsed DNAs and guided by a ssDNA-binding domain. We present a unified model wherein Rad54 acts as an hDNA pump that drives D loop formation while simultaneously removing Rad51 from hDNA, consolidating both ATP-dependent activities of Rad54 into a single mechanistic step.