A Complexed Crystal Structure of a Single-Stranded DNA-Binding Protein with Quercetin and the Structural Basis of Flavonol Inhibition Specificity

Single-stranded DNA (ssDNA)-binding protein (SSB) plays a crucial role in DNA replication, repair, and recombination as well as replication fork restarts. SSB is essential for cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. Whether naturally occurring products can inhibit SSB remains unknown. In this study, the effect of the flavonols myricetin, quercetin, kaempferol, and galangin on the inhibition of Pseudomonas aeruginosa SSB (PaSSB) was investigated. Furthermore, SSB was identified as a novel quercetin-binding protein. Through an electrophoretic mobility shift analysis, myricetin could inhibit the ssDNA binding activity of PaSSB with an IC50 of 2.8 ± 0.4 μM. The effect of quercetin, kaempferol, and galangin was insignificant. To elucidate the flavonol inhibition specificity, the crystal structure of PaSSB complexed with the non-inhibitor quercetin was solved using the molecular replacement method at a resolution of 2.3 Å (PDB entry 7VUM) and compared with a structure with the inhibitor myricetin (PDB entry 5YUN). Although myricetin and quercetin bound PaSSB at a similar site, their binding poses were different. Compared with myricetin, the aromatic ring of quercetin shifted by a distance of 4.9 Å and an angle of 31o for hydrogen bonding to the side chain of Asn108 in PaSSB. In addition, myricetin occupied and interacted with the ssDNA binding sites Lys7 and Glu80 in PaSSB whereas quercetin did not. This result might explain why myricetin could, but quercetin could not, strongly inhibit PaSSB. This molecular evidence reveals the flavonol inhibition specificity and also extends the interactomes of the natural anticancer products myricetin and quercetin to include the OB-fold protein SSB.

The Intrinsically Disordered Region in the Human STN1 OB-Fold Domain Is Important for Protecting Genome Stability

The mammalian CTC1–STN1–TEN1 (CST) complex is an ssDNA-binding protein complex that has emerged as an important player in protecting genome stability and preserving telomere integrity. Studies have shown that CST localizes at stalled replication forks and is critical for protecting the stability of nascent strand DNA. Recent cryo-EM analysis reveals that CST subunits possess multiple OB-fold domains that can form a decameric supercomplex. While considered to be RPA-like, CST acts distinctly from RPA to protect genome stability. Here, we report that while the OB domain of STN1 shares structural similarity with the OB domain of RPA32, the STN1-OB domain contains an intrinsically disordered region (IDR) that is important for maintaining genome stability under replication stress. Single mutations in multiple positions in this IDR, including cancer-associated mutations, cause genome instabilities that are elevated by replication stress and display reduced cellular viability and increased HU sensitivity. While IDR mutations do not impact CST complex formation or CST interaction with its binding partner RAD51, they diminish RAD51 foci formation when replication is perturbed. Interestingly, the IDR is critical for STN1–POLα interaction. Collectively, our results identify the STN1 IDR as an important element in regulating CST function in genome stability maintenance.

Evidence That the Adenovirus Single-Stranded DNA Binding Protein Mediates the Assembly of Biomolecular Condensates to Form Viral Replication Compartments

A common viral replication strategy is characterized by the assembly of intracellular compartments that concentrate factors needed for viral replication and simultaneously conceal the viral genome from host-defense mechanisms. Recently, various membrane-less virus-induced compartments and cellular organelles have been shown to represent biomolecular condensates (BMCs) that assemble through liquid-liquid phase separation (LLPS). In the present work, we analyze biophysical properties of intranuclear replication compartments (RCs) induced during human adenovirus (HAdV) infection. The viral ssDNA-binding protein (DBP) is a major component of RCs that contains intrinsically disordered and low complexity proline-rich regions, features shared with proteins that drive phase transitions. Using fluorescence recovery after photobleaching (FRAP) and time-lapse studies in living HAdV-infected cells, we show that DBP-positive RCs display properties of liquid BMCs, which can fuse and divide, and eventually form an intranuclear mesh with less fluid-like features. Moreover, the transient expression of DBP recapitulates the assembly and liquid-like properties of RCs in HAdV-infected cells. These results are of relevance as they indicate that DBP may be a scaffold protein for the assembly of HAdV-RCs and should contribute to future studies on the role of BMCs in virus-host cell interactions.

Crystal structure of the INTS3/INTS6 complex reveals the functional importance of INTS3 dimerization in DSB repair

SOSS1 is a single-stranded DNA (ssDNA)-binding protein complex that plays a critical role in double-strand DNA break (DSB) repair. SOSS1 consists of three subunits: INTS3, SOSSC, and hSSB1, with INTS3 serving as a scaffold to stabilize this complex. Moreover, the integrator complex subunit 6 (INTS6) participates in the DNA damage response through direct binding to INTS3, but how INTS3 interacts with INTS6, thereby impacting DSB repair, is not clear. Here, we determined the crystal structure of the C-terminus of INTS3 (INTS3c) in complex with the C-terminus of INTS6 (INTS6c) at a resolution of 2.4 Å. Structural analysis revealed that two INTS3c subunits dimerize and interact with INTS6c via conserved residues. Subsequent biochemical analyses confirmed that INTS3c forms a stable dimer and INTS3 dimerization is important for recognizing the longer ssDNA. Perturbation of INTS3c dimerization and disruption of the INTS3c/INTS6c interaction impair the DSB repair process. Altogether, these results unravel the underappreciated role of INTS3 dimerization and the molecular basis of INTS3/INTS6 interaction in DSB repair.

Comparative Genomics of Prophages Sato and Sole Expands the Genetic Diversity Found in the Genus Betatectivirus

Tectiviruses infecting the Bacillus cereus group represent part of the bacterial “plasmid repertoire” as they behave as linear plasmids during their lysogenic cycle. Several novel tectiviruses have been recently found infecting diverse strains belonging the B. cereus lineage. Here, we report and analyze the complete genome sequences of phages Sato and Sole. The linear dsDNA genome of Sato spans 14,852 bp with 32 coding DNA sequences (CDSs), whereas the one of Sole has 14,444 bp comprising 30 CDSs. Both phage genomes contain inverted terminal repeats and no tRNAs. Genomic comparisons and phylogenetic analyses placed these two phages within the genus Betatectivirus in the family Tectiviridae. Additional comparative genomic analyses indicated that the “gene regulation-genome replication” module of phages Sato and Sole is more diverse than previously observed among other fully sequenced betatectiviruses, displaying very low sequence similarities and containing some ORFans. Interestingly, the ssDNA binding protein encoded in this genomic module in phages Sato and Sole has very little amino acid similarity with those of reference betatectiviruses. Phylogenetic analyses showed that both Sato and Sole represent novel tectivirus species, thus we propose to include them as two novel species in the genus Betatectivirus.

The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.

The ssDNA-binding protein MEIOB acts as a dosage-sensitive regulator of meiotic recombination

Meiotic recombination enables reciprocal exchange of genetic information between parental chromosomes and is essential for fertility. MEIOB, a meiosis-specific ssDNA-binding protein, regulates early meiotic recombination. Here we report that the human infertility-associated missense mutation (N64I) in MEIOB causes protein degradation and reduced crossover formation in mouse testes. Although the MEIOB N64I substitution is associated with human infertility, the point mutant mice are fertile despite meiotic defects. Meiob mutagenesis identifies serine 67 as a critical residue for MEIOB. Biochemically, these two mutations (N64I and S67 deletion) cause self-aggregation of MEIOB and sharply reduced protein half-life. Molecular genetic analyses of both point mutants reveal an important role for MEIOB in crossover formation in late meiotic recombination. Furthermore, we find that the MEIOB protein levels directly correlate with the severity of meiotic defects. Our results demonstrate that MEIOB regulates meiotic recombination in a dosage-dependent manner.

Determinants of replication protein A subunit interactions revealed using a phosphomimetic peptide

Replication protein A (RPA) is a eukaryotic ssDNA-binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. However, the structural details and determinants of the phosphorylated RPA32–RPA70 interaction are still unknown. In this study, we provide molecular details of the interaction between RPA70 and a mimic of phosphorylated RPA32 (pmRPA32) using fluorescence polarization and NMR analysis. We show that the N-terminal domain of RPA70 (RPA70N) specifically participates in pmRPA32 binding, whereas the unphosphorylated RPA32 does not bind to RPA70N. Our NMR data revealed that RPA70N binds pmRPA32 using a basic cleft region. We also show that at least 6 negatively charged residues of pmRPA32 are required for RPA70N binding. By introducing alanine mutations into hydrophobic positions of pmRPA32, we found potential points of contact between RPA70N and the N-terminal half of pmRPA32. We used this information to guide docking simulations that suggest the orientation of pmRPA32 in complex with RPA70N. Our study demonstrates detailed features of the domain-domain interaction between RPA70 and RPA32 upon phosphorylation. This result provides insight into how phosphorylation tunes transient bindings between RPA and its partners in DNA resection.

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 theEscherichia coliSSB 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.

PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments

ABSTRACTTranslesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the PCNA sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA damaging agents and is tightly regulated to restrict TLS to damaged DNA. RPA, the major single strand DNA (ssDNA) binding protein, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18•RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination.