Single-stranded Binding Protein Research Articles

Chemical Inhibition of RPA by HAMNO Alters Cell Cycle Dynamics by Impeding DNA Replication and G2-to-M Transition but Has Little Effect on the Radiation-Induced DNA Damage Response.

Replication protein A (RPA) is the major single-stranded DNA (ssDNA) binding protein that is essential for DNA replication and processing of DNA double-strand breaks (DSBs) by homology-directed repair pathways. Recently, small molecule inhibitors have been developed targeting the RPA70 subunit and preventing RPA interactions with ssDNA and various DNA repair proteins. The rationale of this development is the potential utility of such compounds as cancer therapeutics, owing to their ability to inhibit DNA replication that sustains tumor growth. Among these compounds, (1Z)-1-[(2-hydroxyanilino) methylidene] naphthalen-2-one (HAMNO) has been more extensively studied and its efficacy against tumor growth was shown to arise from the associated DNA replication stress. Here, we study the effects of HAMNO on cells exposed to ionizing radiation (IR), focusing on the effects on the DNA damage response and the processing of DSBs and explore its potential as a radiosensitizer. We show that HAMNO by itself slows down the progression of cells through the cell cycle by dramatically decreasing DNA synthesis. Notably, HAMNO also attenuates the progression of G2-phase cells into mitosis by a mechanism that remains to be elucidated. Furthermore, HAMNO increases the fraction of chromatin-bound RPA in S-phase but not in G2-phase cells and suppresses DSB repair by homologous recombination. Despite these marked effects on the cell cycle and the DNA damage response, radiosensitization could neither be detected in exponentially growing cultures, nor in cultures enriched in G2-phase cells. Our results complement existing data on RPA inhibitors, specifically HAMNO, and suggest that their antitumor activity by replication stress induction may not extend to radiosensitization. However, it may render cells more vulnerable to other forms of DNA damaging agents through synthetically lethal interactions, which requires further investigation.

Crystal Structure of DNA Replication Protein SsbA Complexed with the Anticancer Drug 5-Fluorouracil.

Single-stranded DNA-binding proteins (SSBs) play a crucial role in DNA metabolism by binding and stabilizing single-stranded DNA (ssDNA) intermediates. Through their multifaceted roles in DNA replication, recombination, repair, replication restart, and other cellular processes, SSB emerges as a central player in maintaining genomic integrity. These attributes collectively position SSBs as essential guardians of genomic integrity, establishing interactions with an array of distinct proteins. Unlike Escherichia coli, which contains only one type of SSB, some bacteria have two paralogous SSBs, referred to as SsbA and SsbB. In this study, we identified Staphylococcus aureus SsbA (SaSsbA) as a fresh addition to the roster of the anticancer drug 5-fluorouracil (5-FU) binding proteins, thereby expanding the ambit of the 5-FU interactome to encompass this DNA replication protein. To investigate the binding mode, we solved the complexed crystal structure with 5-FU at 2.3 Å (PDB ID 7YM1). The structure of glycerol-bound SaSsbA was also determined at 1.8 Å (PDB ID 8GW5). The interaction between 5-FU and SaSsbA was found to involve R18, P21, V52, F54, Q78, R80, E94, and V96. Based on the collective results from mutational and structural analyses, it became evident that SaSsbA's mode of binding with 5-FU diverges from that of SaSsbB. This complexed structure also holds the potential to furnish valuable comprehension regarding how 5-FU might bind to and impede analogous proteins in humans, particularly within cancer-related signaling pathways. Leveraging the information furnished by the glycerol and 5-FU binding sites, the complexed structures of SaSsbA bring to the forefront the potential viability of several interactive residues as potential targets for therapeutic interventions aimed at curtailing SaSsbA activity. Acknowledging the capacity of microbiota to influence the host's response to 5-FU, there emerges a pressing need for further research to revisit the roles that bacterial and human SSBs play in the realm of anticancer therapy.

Xenopus Ssbp2 is required for embryonic pronephros morphogenesis and terminal differentiation

The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary conservation of gene expression and segmentation patterning between mammalian and amphibian nephrons, the Xenopus laevis pronephric kidney offers a simplified model for studying nephrogenesis. The Lhx1 transcription factor plays several roles during embryogenesis, regulating target genes expression by forming multiprotein complexes with LIM binding protein 1 (Ldb1). However, few Lhx1-Ldb1 cofactors have been identified for kidney organogenesis. By tandem- affinity purification from kidney-induced Xenopus animal caps, we identified single-stranded DNA binding protein 2 (Ssbp2) interacts with the Ldb1–Lhx1 complex. Ssbp2 is expressed in the Xenopus pronephros, and knockdown prevents normal morphogenesis and differentiation of the glomus and the convoluted renal tubules. We demonstrate a role for a member of the Ssbp family in kidney organogenesis and provide evidence of a fundamental function for the Ldb1–Lhx1-Ssbp transcriptional complexes in embryonic development.

Application of recombinant human pyruvate kinase in recombinase polymerase amplification

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells. RPA with PKM was performed at 41°C with the invitro synthesized urease subunit β (ureB) DNA from Ureaplasma parvum serovar 3 as a standard DNA. The optimal concentrations of PKM and phosphoenolpyruvate were 20ng/μL and 10mM, respectively. The RPA reaction with PKM was more sensitive than that with CK. PKM exhibited higher thermostability than CK, suggesting that the RPA reagents with PKM are preferable to those with CK for onsite use.

Interaction of a Dimeric Single-Stranded DNA-Binding Protein (G5P) with DNA Hairpins. A Molecular Beacon Study.

Gene-V protein (G5P/GVP) is a single-stranded (ss)DNA-binding protein (SBP) of bacteriophage f1 that is required for DNA synthesis and repair. In solution, it exists as a dimer that binds two antiparallel ssDNA strands with high affinity in a cooperative manner, forming a left-handed helical protein-DNA filament. Here, we report on fluorescence studies of the interaction of G5P with different DNA oligonucleotides having a hairpin structure (molecular beacon, MB) with a seven base-pair stem (dT24-stem7, dT18-stem7), as well as with DNA oligonucleotides (dT38, dT24) without a defined secondary structure. All oligonucleotides were end-labeled with a Cy3-fluorophore and a BHQ2-quencher. In the case of DNA oligonucleotides without a secondary structure, an almost complete quenching of their strong fluorescence (with about 5% residual intensity) was observed upon the binding of G5P. This implies an exact alignment of the ends of the DNA strand(s) in the saturated complex. The interaction of the DNA hairpins with G5P led to the unzipping of the base-paired stem, as revealed by fluorescence measurements, fluorescence microfluidic mixing experiments, and electrophoretic mobility shift assay data. Importantly, the disruption of ssDNA's secondary structure agrees with the behavior of other single-stranded DNA-binding proteins (SBPs). In addition, substantial protein-induced fluorescence enhancement (PIFE) of the Cy3-fluorescence was observed.

Structural characterization of human RPA70N association with DNA damage response proteins.

The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA metabolism, especially DNA damage response. The N-terminal OB domain of the RPA70 subunit (RPA70N) is a major protein-protein interaction element for RPA and binds to more than 20 partner proteins. Previous crystallography studies of RPA70N with p53, DNA2 and PrimPol fragments revealed that RPA70N binds to amphipathic peptides that mimic ssDNA. NMR chemical-shift studies also provided valuable information on the interaction of RPA70N residues with target sequences. However, it is still unclear how RPA70N recognizes and distinguishes such a diverse group of target proteins. Here, we present high-resolution crystal structures of RPA70N in complex with peptides from eight DNA damage response proteins. The structures show that, in addition to the ssDNA mimicry mode of interaction, RPA70N employs multiple ways to bind its partners. Our results advance the mechanistic understanding of RPA70N-mediated recruitment of DNA damage response proteins.

Advances in the application of recombinase-aided amplification combined with CRISPR-Cas technology in quick detection of pathogenic microbes.

The rapid diagnosis of pathogenic infections plays a vital role in disease prevention, control, and public health safety. Recombinase-aided amplification (RAA) is an innovative isothermal nucleic acid amplification technology capable of fast DNA or RNA amplification at low temperatures. RAA offers advantages such as simplicity, speed, precision, energy efficiency, and convenient operation. This technology relies on four essential components: recombinase, single-stranded DNA-binding protein (SSB), DNA polymerase, and deoxyribonucleoside triphosphates, which collectively replace the laborious thermal cycling process of traditional polymerase chain reaction (PCR). In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) system, a groundbreaking genome engineering tool, has garnered widespread attention across biotechnology, agriculture, and medicine. Increasingly, researchers have integrated the recombinase polymerase amplification system (or RAA system) with CRISPR technology, enabling more convenient and intuitive determination of detection results. This integration has significantly expanded the application of RAA in pathogen detection. The step-by-step operation of these two systems has been successfully employed for molecular diagnosis of pathogenic microbes, while the single-tube one-step method holds promise for efficient pathogen detection. This paper provides a comprehensive review of RAA combined with CRISPR-Cas and its applications in pathogen detection, aiming to serve as a valuable reference for further research in related fields.

SOSSB1 and SOSSB2 mutually regulate protein stability through competitive binding of SOSSA

Human single-stranded DNA-binding protein homologs hSSB1 (SOSSB1) and hSSB2 (SOSSB2) make a vital impact on maintaining genome stability as the B subunits of the sensor of single-stranded DNA complex (SOSS). However, whether and how SOSSB1 and SOSSB2 modulate mutual expression is unclear. This study, demonstrated that the depletion of SOSSB1 in cells enhances the stability of the SOSSB2 protein, and conversely, SOSSB2 depletion enhances the stability of the SOSSB1 protein. The levels of SOSSB1 and SOSSB2 proteins are mutually regulated through their competitive binding with SOSSA which associates with the highly conservative OB-fold domain in SOSSB1 and SOSSB2. The destabilized SOSSB1 and SOSSB2 proteins can be degraded via the proteasome pathway. Additionally, the simultaneous loss of SOSSB1 and SOSSB2 aggravates homologous recombination (HR)-mediated DNA repair defects, enhances cellular radiosensitivity and promotes cell apoptosis. In conclusion, in this study, we showed that SOSSB1 and SOSSB2 positively regulate HR repair and the interaction between SOSSA and SOSSB1 or SOSSB2 prevents the degradation of SOSSB1 and SOSSB2 proteins via the proteasome pathway.

Precise genome engineering in Pseudomonas using phage-encoded homologous recombination and the Cascade-Cas3 system.

A lack of generic and effective genetic manipulation methods for Pseudomonas has restricted fundamental research and utilization of this genus for biotechnology applications. Phage-encoded homologous recombination (PEHR) is an efficient tool for bacterial genome engineering. This PEHR system is based on a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (Rec-TEPsy) from P. syringae pv. syringae B728a and also contains exogenous elements, including the RecBCD inhibitor (Redγ or Pluγ) or single-stranded DNA-binding protein (SSB), that were added to enhance the PEHR recombineering efficiency. To solve the problem of false positives in Pseudomonas editing with the PEHR system, the processive enzyme Cas3 with a minimal Type I-C Cascade-based system was combined with PEHR. This protocol describes the utilization of a Pseudomonas-specific PEHR-Cas3 system that was designed to universally and proficiently modify the genomes of Pseudomonas species. The pipeline uses standardized cassettes combined with the concerted use of SacB counterselection and Cre site-specific recombinase for markerless or seamless genome modification, in association with vectors that possess the selectively replicating template R6K to minimize recombineering background. Compared with the traditional allelic exchange editing method, the PEHR-Cas3 system does not need to construct suicide plasmids carrying long homologous arms, thus simplifying the experimental procedure and shortening the traceless editing period. Compared with general editing systems based on phage recombinases, the PEHR-Cas3 system can effectively improve the screening efficiency of mutants using the cutting ability of Cas3 protein. The entire procedure requires ~12 days.

DNA damage alters binding conformations of E. coli single-stranded DNA-binding protein

Single-stranded DNA-binding proteins (SSBs) are essential cellular components, binding to transiently exposed regions of single-stranded DNA (ssDNA) with high affinity and sequence non-specificity to coordinate DNA repair and replication. Escherichia coli SSB (EcSSB) is a homotetramer that wraps variable lengths of ssDNA in multiple conformations (typically occupying either 65 or 35 nt), which is well studied across experimental conditions of substrate length, salt, pH, temperature, etc. In this work, we use atomic force microscopy to investigate the binding of SSB to individual ssDNA molecules. We introduce non-canonical DNA bases that mimic naturally occurring DNA damage, synthetic abasic sites, as well as a non-DNA linker into our experimental constructs at sites predicted to interact with EcSSB. By measuring the fraction of DNA molecules with EcSSB bound as well as the volume of protein bound per DNA molecule, we determine the protein binding affinity, cooperativity, and conformation. We find that, with only one damaged nucleotide, the binding of EcSSB is unchanged relative to its binding to undamaged DNA. In the presence of either two tandem abasic sites or a non-DNA spacer, however, the binding affinity associated with a single EcSSB tetramer occupying the full substrate in the 65-nt mode is greatly reduced. In contrast, the binding of two EcSSB tetramers, each in the 35-nt mode, is preserved. Changes in the binding and cooperative behaviors of EcSSB across these constructs can inform how genomic repair and replication processes may change as environmental damage accumulates in DNA.

Sanguinarine induces apoptosis in osteosarcoma by attenuating the binding of STAT3 to the single-stranded DNA-binding protein 1 (SSBP1) promoter region.

Osteosarcoma, a primary malignant bone tumour prevalent among adolescents and young adults, remains a considerable challenge despite protracted progress made in enhancing patient survival rates over the last 40 years. Consequently, the development of novel therapeutic approaches for osteosarcoma is imperative. Sanguinarine (SNG), a compound with demonstrated potent anticancer properties against various malignancies, presents a promising avenue for exploration. Nevertheless, the intricate molecular mechanisms underpinning SNG's actions in osteosarcoma remain elusive, necessitating further elucidation. Single-stranded DNA-binding protein 1 (SSBP1) was screened out by differential proteomic analysis. Apoptosis, cell cycle, reactive oxygen species (ROS) and mitochondrial changes were assessed via flow cytometry. Western blotting and quantitative real-time reverse transcription PCR (qRT-PCR) were used to determine protein and gene levels. The antitumour mechanism of SNG was explored at a molecular level using chromatin immunoprecipitation (ChIP) and dual luciferase reporter plasmids. Our investigation revealed that SNG exerted an up-regulated effect on SSBP1, disrupting mitochondrial function and inducing apoptosis. In-depth analysis uncovered a mechanism whereby SNG hindered the JAK/signal transducer and activator of transcription 3 (STAT3) signalling pathway, relieved the inhibitory effect of STAT3 on SSBP1 transcription, and inhibited the downstream PI3K/Akt/mTOR signalling axis, ultimately activating apoptosis. The study delved further into elucidating the anticancer mechanism of SNG in osteosarcoma. Notably, we unravelled the previously undisclosed apoptotic potential of SSBP1 in osteosarcoma cells. This finding holds substantial promise in advancing the development of novel anticancer drugs and identification of therapeutic targets.

The CST complex facilitates cell survival under oxidative genotoxic stress.

Genomic DNA is constantly exposed to a variety of genotoxic stresses, and it is crucial for organisms to be equipped with mechanisms for repairing the damaged genome. Previously, it was demonstrated that the mammalian CST (CTC1-STN1-TEN1) complex, which was originally identified as a single-stranded DNA-binding trimeric protein complex essential for telomere maintenance, is required for survival in response to hydroxyurea (HU), which induces DNA replication fork stalling. It is still unclear, however, how the CST complex is involved in the repair of diverse types of DNA damage induced by oxidizing agents such as H2O2. STN1 knockdown (KD) sensitized HeLa cells to high doses of H2O2. While H2O2 induced DNA strand breaks throughout the cell cycle, STN1 KD cells were as resistant as control cells to H2O2 treatment when challenged in the G1 phase of the cell cycle, but they were sensitive when exposed to H2O2 in S/G2/M phase. STN1 KD cells showed a failure of DNA synthesis and RAD51 foci formation upon H2O2 treatment. Chemical inhibition of RAD51 in shSTN1 cells did not exacerbate the sensitivity to H2O2, implying that the CST complex and RAD51 act in the same pathway. Collectively, our results suggest that the CST complex is required for maintaining genomic stability in response to oxidative DNA damage, possibly through RAD51-dependent DNA repair/protection mechanisms.

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.

G-quadruplex resolution: From molecular mechanisms to physiological relevance

Guanine-rich DNA sequences can fold into stable four-stranded structures called G-quadruplexes or G4s. Research in the past decade demonstrated that G4 structures are widespread in the genome and prevalent in regulatory regions of actively transcribed genes. The formation of G4s has been tightly linked to important biological processes including regulation of gene expression and genome maintenance. However, they can also pose a serious threat to genome integrity especially by impeding DNA replication, and G4-associated somatic mutations have been found accumulated in the cancer genomes. Specialised DNA helicases and single stranded DNA binding proteins that can resolve G4 structures play a crucial role in preventing genome instability. The large variety of G4 unfolding proteins suggest the presence of multiple G4 resolution mechanisms in cells. Recently, there has been considerable progress in our detailed understanding of how G4s are resolved, especially during DNA replication. In this review, we first discuss the current knowledge of the genomic G4 landscapes and the impact of G4 structures on DNA replication and genome integrity. We then describe the recent progress on the mechanisms that resolve G4 structures and their physiological relevance. Finally, we discuss therapeutic opportunities to target G4 structures.

The SSBP3 co-regulator is required for glucose homeostasis, pancreatic islet architecture, and beta-cell identity

ObjectiveTranscriptional complex activity drives the development and function of pancreatic islet cells to allow for proper glucose regulation. Prior studies from our lab and others highlighted that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), and its interacting co-regulator, Ldb1, are vital effectors of developing and adult β-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with Isl1 and Ldb1 in β-cells and primary islets (mouse and human) to impact β-cell target genes MafA and Glp1R in vitro. Members of the SSBP family stabilize TF complexes by binding directly to Ldb1 and protecting the complex from ubiquitin-mediated turnover. In this study, we hypothesized that SSBP3 has critical roles in pancreatic islet cell function in vivo, similar to the Isl1::Ldb1 complex. MethodsWe first developed a novel SSBP3 LoxP allele mouse line, where Cre-mediated recombination imparts a predicted early protein termination. We bred this mouse with constitutive Cre lines (Pdx1- and Pax6-driven) to recombine SSBP3 in the developing pancreas and islet (SSBP3ΔPanc and SSBP3ΔIslet), respectively. We assessed glucose tolerance and used immunofluorescence to detect changes in islet cell abundance and markers of β-cell identity and function. Using an inducible Cre system, we also deleted SSBP3 in the adult β-cell, a model termed SSBP3Δβ-cell. We measured glucose tolerance as well as glucose-stimulated insulin secretion (GSIS), both in vivo and in isolated islets in vitro. Using islets from control and SSBP3Δβ-cell we conducted RNA-Seq and compared our results to published datasets for similar β-cell specific Ldb1 and Isl1 knockouts to identify commonly regulated target genes. ResultsSSBP3ΔPanc and SSBP3ΔIslet neonates present with hyperglycemia. SSBP3ΔIslet mice are glucose intolerant by P21 and exhibit a reduction of β-cell maturity markers MafA, Pdx1, and UCN3. We observe disruptions in islet cell architecture with an increase in glucagon+ α-cells and ghrelin+ ε-cells at P10. Inducible loss of β-cell SSBP3 in SSBP3Δβ-cell causes hyperglycemia, glucose intolerance, and reduced GSIS. Transcriptomic analysis of 14-week-old SSBP3Δβ-cell islets revealed a decrease in β-cell function gene expression (Ins, MafA, Ucn3), increased stress and dedifferentiation markers (Neurogenin-3, Aldh1a3, Gastrin), and shared differentially expressed genes between SSBP3, Ldb1, and Isl1 in adult β-cells. ConclusionsSSBP3 drives proper islet identity and function, where its loss causes altered islet-cell abundance and glucose homeostasis. β-Cell SSBP3 is required for GSIS and glucose homeostasis, at least partially through shared regulation of Ldb1 and Isl1 target genes.

The ortholog of human ssDNA-binding protein SSBP3 influences neurodevelopment and autism-like behaviors in Drosophila melanogaster.

1p32.3 microdeletion/duplication is implicated in many neurodevelopmental disorders-like phenotypes such as developmental delay, intellectual disability, autism, macro/microcephaly, and dysmorphic features. The 1p32.3 chromosomal region harbors several genes critical for development; however, their validation and characterization remain inadequate. One such gene is the single-stranded DNA-binding protein 3 (SSBP3) and its Drosophila melanogaster ortholog is called sequence-specific single-stranded DNA-binding protein (Ssdp). Here, we investigated consequences of Ssdp manipulations on neurodevelopment, gene expression, physiological function, and autism-associated behaviors using Drosophila models. We found that SSBP3 and Ssdp are expressed in excitatory neurons in the brain. Ssdp overexpression caused morphological alterations in Drosophila wing, mechanosensory bristles, and head. Ssdp manipulations also affected the neuropil brain volume and glial cell number in larvae and adult flies. Moreover, Ssdp overexpression led to differential changes in synaptic density in specific brain regions. We observed decreased levels of armadillo in the heads of Ssdp overexpressing flies, as well as a decrease in armadillo and wingless expression in the larval wing discs, implicating the involvement of the canonical Wnt signaling pathway in Ssdp functionality. RNA sequencing revealed perturbation of oxidative stress-related pathways in heads of Ssdp overexpressing flies. Furthermore, Ssdp overexpressing brains showed enhanced reactive oxygen species (ROS), altered neuronal mitochondrial morphology, and up-regulated fission and fusion genes. Flies with elevated levels of Ssdp exhibited heightened anxiety-like behavior, altered decisiveness, defective sensory perception and habituation, abnormal social interaction, and feeding defects, which were phenocopied in the pan-neuronal Ssdp knockdown flies, suggesting that Ssdp is dosage sensitive. Partial rescue of behavioral defects was observed upon normalization of Ssdp levels. Notably, Ssdp knockdown exclusively in adult flies did not produce behavioral and functional defects. Finally, we show that optogenetic manipulation of Ssdp-expressing neurons altered autism-associated behaviors. Collectively, our findings provide evidence that Ssdp, a dosage-sensitive gene in the 1p32.3 chromosomal region, is associated with various anatomical, physiological, and behavioral defects, which may be relevant to neurodevelopmental disorders like autism. Our study proposes SSBP3 as a critical gene in the 1p32.3 microdeletion/duplication genomic region and sheds light on the functional role of Ssdp in neurodevelopmental processes in Drosophila.

Dynamic structure of T4 gene 32 protein filaments facilitates rapid noncooperative protein dissociation.

Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication. gp32 forms cooperative filaments on ssDNA through interprotein interactions between its core and N-terminus. However, detailed understanding of gp32 filament structure and organization remains incomplete, particularly for longer, biologically-relevant DNA lengths. Moreover, it is unclear how these tightly-bound filaments dissociate from ssDNA during complementary strand synthesis. We use optical tweezers and atomic force microscopy to probe the structure and binding dynamics of gp32 on long (∼8 knt) ssDNA substrates. We find that cooperative binding of gp32 rigidifies ssDNA while also reducing its contour length, consistent with the ssDNA helically winding around the gp32 filament. While measured rates of gp32 binding and dissociation indicate nM binding affinity, at ∼1000-fold higher protein concentrations gp32 continues to bind into and restructure the gp32-ssDNA filament, leading to an increase in its helical pitch and elongation of the substrate. Furthermore, the oversaturated gp32-ssDNA filament becomes progressively unwound and unstable as observed by the appearance of a rapid, noncooperative protein dissociation phase not seen at lower complex saturation, suggesting a possible mechanism for prompt removal of gp32 from the overcrowded ssDNA in front of the polymerase during replication.

Alteration of replication protein A binding mode on single-stranded DNA by NSMF potentiates RPA phosphorylation by ATR kinase.

Replication protein A (RPA), a eukaryotic single-stranded DNA (ssDNA) binding protein, dynamically interacts with ssDNA in different binding modes and plays essential roles in DNA metabolism such as replication, repair, and recombination. RPA accumulation on ssDNA due to replication stress triggers the DNA damage response (DDR) by activating the ataxia telangiectasia and RAD3-related (ATR) kinase, which phosphorylates itself and downstream DDR factors, including RPA. We recently reported that the N-methyl-D-aspartate receptor synaptonuclear signaling and neuronal migration factor (NSMF), a neuronal protein associated with Kallmann syndrome, promotes RPA32 phosphorylation via ATR upon replication stress. However, how NSMF enhances ATR-mediated RPA32 phosphorylation remains elusive. Here, we demonstrate that NSMF colocalizes and physically interacts with RPA at DNA damage sites in vivo and in vitro. Using purified RPA and NSMF in biochemical and single-molecule assays, we find that NSMF selectively displaces RPA in the more weakly bound 8- and 20-nucleotide binding modes from ssDNA, allowing the retention of more stable RPA molecules in the 30-nt binding mode. The 30-nt binding mode of RPA enhances RPA32 phosphorylation by ATR, and phosphorylated RPA becomes stabilized on ssDNA. Our findings provide new mechanistic insight into how NSMF facilitates the role of RPA in the ATR pathway.

Immunohistochemical Analysis of Single-Stranded DNA Binding Protein 2 in Non-Melanoma Skin Cancers.

Single-stranded DNA binding protein 2 (SSBP2) is a tumor suppressor candidate. In this study, the expression level and clinicopathological significance of SSBP2 in squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) were evaluated. We also identified biological pathways associated with a set of genes potentially related to SSBP2. Immunohistochemistry (IHC) was performed on 70 SCC and 146 BCC cases to assess SSBP2 expression semi-quantitatively. In addition, the associations between SSBP2 expression and clinicopathological characteristics were analyzed. Gene ontology (GO) enrichment analysis was performed using publicly available data and web-based bioinformatics tools. Compared with BCC, SCC had a significantly low SSBP2 expression (p < 0.001). In total, 12 (17.1%) of the 70 SCC cases and 30 (20.5%) of the 146 BCC cases showed low SSBP2 expression. Among SCC cases, ulceration (p = 0.005) and a deep level of invasion (p = 0.012) showed an association with low SSBP2 expression. Local recurrence was slightly more common in the SCC subgroup with low SSBP2 expression, although the difference was not significant (p = 0.058). Using GO enrichment analysis, we identified several biological functions performed by a set of 36 genes in SCC. SSBP2 evaluation using IHC can be helpful in the differential diagnosis of SCC and BCC. SSBP2 expression was associated with tumor invasiveness in SCC.

Structural basis of the interaction between BCL9-Pygo and LDB-SSBP complexes in assembling the Wnt enhanceosome

The Wnt enhanceosome is responsible for transactivation of Wnt-responsive genes and a promising therapeutic target for treatment of numerous cancers with Adenomatous Polyposis Coli (APC) or β-catenin mutations. How the Wnt enhanceosome is assembled remains poorly understood. Here we show that B-cell lymphoma 9 protein (BCL9), Pygopus (Pygo), LIM domain-binding protein 1 (LDB1) and single-stranded DNA-binding protein (SSBP) form a stable core complex within the Wnt enhanceosome. Their mutual interactions rely on a highly conserved N-terminal asparagine proline phenylalanine (NPF) motif of Pygo, through which the BCL9-Pygo complex binds to the LDB-SSBP core complex. Our crystal structure of a ternary complex comprising the N-terminus of human Pygo2, LDB1 and SSBP2 reveals a single LDB1-SSBP2 complex binding simultaneously to two Pygo2 molecules via their NPF motifs. These interactions critically depend on the NPF motifs which bind to a deep groove formed between LDB1 and SSBP2, potentially constituting a binding site for drugs blocking Wnt/β-catenin signaling. Analysis of human cell lines lacking LDB or Pygo supports the functional relevance of the Pygo-LDB1-SSBP2 interaction for Wnt/β-catenin-dependent transcription.