Ja Yil Lee

Defined DNA lesions are essential substrates for dissecting DNA repair mechanisms, yet existing approaches remain limited in positional flexibility, lesion diversity, efficiency, or compatibility with long and chromatinized DNA. Here, we present CGRID (CRISPR-mediated Gap-based Replacement for Insertion of DNA damage), a programmable strategy for site-specific installation of DNA lesions into long DNA and nucleosomes. CGRID uses paired Cas9 nickases to generate two adjacent nicks, followed by gap formation and replacement with a synthetic lesion-containing oligonucleotide. This design enables easy and efficient insertion of diverse DNA lesions at user-defined positions without pre-engineered nicking sites or substrate redesign. Applied to long λ-DNA, CGRID supports efficient installation of single lesions and internal double-strand breaks at predefined positions, which are challenging to obtain using existing methods. These molecules enable high-throughput single-molecule analysis of diverse repair pathways including base excision repair (BER), nonhomologous end joining, and microhomology-mediated end joining without hydrodynamic force. CGRID further allows efficient site-specific lesion incorporation into nucleosomal DNA, enabling systematic repositioning of damage within a nucleosome. This shows that nucleosome context and lesion orientation strongly influence APE1 incision efficiency. Together, CGRID establishes a broadly applicable platform for programmable DNA damage engineering, bridging biochemical and single-molecule methodologies, and facilitates mechanistic studies of chromatin-dependent genome maintenance.

The online version contains supplementary material available at 10.1186/s40580-026-00556-y.

Efficient recognition of DNA lesions such as apurinic/apyrimidinic (AP) sites is essential for maintaining genome stability. Apurinic/apyrimidinic endonuclease 1 (APE1) is the primary eukaryotic AP endonuclease, yet how it identifies rare lesions among vast stretches of undamaged DNA remains incompletely understood. Using single-molecule imaging combined with molecular dynamics simulations, we reveal that APE1 employs a distinctive dual mechanism to search DNA damage. First, Mg2+ coordination at the active site neutralizes clustered negative charges, stabilizing electrostatic contacts during scanning. Second, its N-terminal intrinsically disordered region (IDR)—a feature conserved only in eukaryotic homologs but absent in prokaryotic ExoIII—not only interacts with DNA through transient IDR contacts but also engages continuous interactions via the unprecedented Arg177 residue within the structured nuclease domain, thereby prolonging residence time and enabling long-range diffusion. Together, these two modules synergize to promote a sliding-based search strategy tailored to the complexity of eukaryotic genomes. Consistent with this model, IDR deletion restricts APE1 to 3D collisions, whereas IDR duplication enhances 1D scanning. Thus, APE1 exemplifies how structural disorder and metal-ion coordination integrate to enable long-range lesion recognition, highlighting an evolutionary innovation in eukaryotic DNA repair.

Quantum dots (QDs) are fluorescent nanoparticles widely used for single-molecule imaging because of their exceptional brightness and photostability. However, the impact of QD surface chemistry on biomolecular interactions has not been systematically investigated. Here, we report that commercial QDs unexpectedly destabilize protein-DNA complexes by inducing protein dissociation from DNA. Using the human nucleotide excision repair protein, xeroderma pigmentosum complementation group A (XPA) as a model system, we demonstrate that antibody-conjugated QDs promote dissociation of XPA from DNA substrates, independently of sizes and surface modification of QDs, antibody types, epitope tags, buffer conditions, or DNA structures. We find that polyethylene glycol (PEG), a common polymer coating on QD surfaces, is the primary factor responsible for this effect. To tackle this problem, we engineered QDs with precisely controlled surface polymer compositions. By systematically changing the ratio of anchoring, hydrophilic, and PEG-based functional groups, we find that reducing PEG density below a critical threshold effectively suppresses protein dissociation while maintaining excellent colloidal stability and brightness. Furthermore, antibodies conjugated via click chemistry between azido groups and DBCO enabled specific labeling of XPA without perturbing the DNA binding activity. Using these optimized QDs, we conducted single-molecule DNA curtain assays to visualize XPA-DNA interactions. QD-labeled XPA exhibits one-dimensional diffusion with frequent pausing on undamaged DNA. DNA curtain assays revealed that XPA preferentially binds DNA bubbles and searches for bubble structures through both one-dimensional diffusion and three-dimensional collision. Quantitative analysis showed that three-dimensional collision is the dominant pathway for bubble recognition. Taken together, our results uncover a previously unrecognized limitation of PEG-coated QDs in single-molecule studies and provide a general surface-engineering strategy to preserve native protein-DNA interactions. Newly engineered QDs establish robust platforms for accurate single-molecule visualization of biomolecular processes.

Ultraviolet-induced DNA lesions are removed by the nucleotide excision repair (NER) pathway. In global-genome NER (GG-NER), XPC-RAD23B recognizes the lesions and initiates NER. However, cyclobutane pyrimidine dimers (CPDs), which do not significantly destabilize the DNA duplex, are not bound by XPC-RAD23B with high selectivity. Instead, CPD is preferentially sensed by UV-DDB, which is believed to hand over the lesion to XPC-RAD23B via ubiquitination of both proteins. Here, by combining biochemical and single-molecule DNA curtain assays, we investigate the interactions between UV-DDB and XPC-RAD23B on DNA. Surprisingly, we discover that XPC-RAD23B enhances the binding of UV-DDB to DNA. We demonstrate that this enhancement can be attributed to the complex formation of UV-DDB and XPC-RAD23B (UX-complex), which increases the binding affinity of UV-DDB to undamaged DNA. We further show that UV-DDB finds CPDs through one-dimensional (1D) diffusion along DNA. Collectively, the UX-complex enhances UV-DDB loading to DNA to accelerate the search for CPD via 1D diffusion. Moreover, we find that UV-DDB and XPC-RAD23B can bind CPDs as a complex, which facilitates the transfer of CPD. Altogether, our results show that UV-DDB and XPC-RAD23B cooperatively interact for rapid CPD search, providing a new mechanism for lesion search in GG-NER.

The histone chaperone FAcilitates Chromatin Transcription (FACT) is a heterodimeric complex consisting of Spt16 and Pob3, crucial for preserving nucleosome integrity during transcription and DNA replication. Loss of FACT leads to cryptic transcription and heterochromatin defects. FACT was shown to interact with Abo1, an AAA + family histone chaperone involved in nucleosome dynamics. Depletion of Abo1 causes FACT to stall at transcription start sites and mimics FACT mutants, indicating a functional association between Abo1 and FACT. However, the precise role of Abo1 in FACT function remains poorly understood. Here, we reveal that Abo1 directly interacts with FACT and facilitates the dissociation of FACT from nucleosome. Specifically, the N-terminal region of Abo1 utilizes its FACT-interacting helix to bind to the N-terminal domain of Spt16. In addition, using single-molecule fluorescence imaging, we discovered that Abo1 facilitates the ATP-dependent dissociation of FACT from nucleosomes. Furthermore, we demonstrate that the interaction between Abo1 and FACT is essential for maintaining heterochromatin in fission yeast. In summary, our findings suggest that Abo1 regulates FACT turnover in an ATP-dependent manner, proposing a model of histone chaperone recycling driven by inter-chaperone interactions.

The histone chaperone FACT is a heterodimeric complex consisting of Spt16 and Pob3, crucial for preserving nucleosome integrity during transcription and DNA replication. Loss of FACT leads to cryptic transcription and heterochromatin defects. FACT was shown to interact with Abo1, an AAA+ family histone chaperone involved in nucleosome dynamics. Depletion of Abo1 causes FACT to stall at transcription start sites (TSS) and mimics FACT mutants, indicating a functional association between Abo1 and FACT. However, the precise role of Abo1 in FACT function remains poorly understood. Here, we reveal that Abo1 directly interacts with FACT and facilitates the dissociation of FACT from chromatin. Specifically, the N-terminal region of Abo1 utilizes its FACT interacting (FIN) helix to bind to the N-terminal domain (NTD) of Spt16. In addition, using single-molecule fluorescence imaging, we discovered that Abo1 facilitates the ATP-dependent dissociation of FACT from nucleosomes. Furthermore, we demonstrate that the interaction between Abo1 and FACT is essential for maintaining heterochromatin in fission yeast. In summary, our findings suggest that Abo1 regulates FACT turnover in an ATP-dependent manner, proposing a model of histone chaperone recycling driven by inter-chaperone interactions.