Jinglong Wang

All Publications

• Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation. Nature structural & molecular biology Wang, J., Le Gall, J., Frock, R. L., Strick, T. R. 2023

  Abstract

  Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3' overhangs can be robustly generated via a 2-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends.

  View details for DOI 10.1038/s41594-023-01104-6

  View details for PubMedID 37828409

  View details for PubMedCentralID 5898235

• Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation BioRxiv Wang, J. 2022

  View details for DOI 10.1101/2022.11.08.515552

• Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis. Nucleic acids research Öz, R. n., Wang, J. L., Guerois, R. n., Goyal, G. n., Kk, S. n., Ropars, V. n., Sharma, R. n., Koca, F. n., Charbonnier, J. B., Modesti, M. n., Strick, T. R., Westerlund, F. n. 2021

  Abstract

  We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.

  View details for DOI 10.1093/nar/gkab083

  View details for PubMedID 33590005

• Mechanism of efficient double-strand break repair by a long non-coding RNA. Nucleic acids research Thapar, R., Wang, J. L., Hammel, M., Ye, R., Liang, K., Sun, C., Hnizda, A., Liang, S., Maw, S. S., Lee, L., Villarreal, H., Forrester, I., Fang, S., Tsai, M. S., Blundell, T. L., Davis, A. J., Lin, C., Lees-Miller, S. P., Strick, T. R., Tainer, J. A. 2020

  Abstract

  Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

  View details for DOI 10.1093/nar/gkaa784

  View details for PubMedID 33045735

• A Modular DNA Scaffold to Study Protein-Protein Interactions at Single-Molecule Resolution Kostrz, D. N., Wayment-Steele, H. K., Wang, J., Follenfant, M., Pande, V. S., Triller, A., Specht, C. G., Strick, T. R., Gosse, C. CELL PRESS. 2020: 187A

  View details for Web of Science ID 000513023201184

• A modular DNA scaffold to study protein-protein interactions at single-molecule resolution. Nature nanotechnology Kostrz, D. n., Wayment-Steele, H. K., Wang, J. L., Follenfant, M. n., Pande, V. S., Strick, T. R., Gosse, C. n. 2019

  Abstract

  The residence time of a drug on its target has been suggested as a more pertinent metric of therapeutic efficacy than the traditionally used affinity constant. Here, we introduce junctured-DNA tweezers as a generic platform that enables real-time observation, at the single-molecule level, of biomolecular interactions. This tool corresponds to a double-strand DNA scaffold that can be nanomanipulated and on which proteins of interest can be engrafted thanks to widely used genetic tagging strategies. Thus, junctured-DNA tweezers allow a straightforward and robust access to single-molecule force spectroscopy in drug discovery, and more generally in biophysics. Proof-of-principle experiments are provided for the rapamycin-mediated association between FKBP12 and FRB, a system relevant in both medicine and chemical biology. Individual interactions were monitored under a range of applied forces and temperatures, yielding after analysis the characteristic features of the energy profile along the dissociation landscape.

  View details for DOI 10.1038/s41565-019-0542-7

  View details for PubMedID 31548690

• Dissection of DNA double-strand-break repair using novel single-molecule forceps. Nature structural & molecular biology Wang, J. L., Duboc, C., Wu, Q., Ochi, T., Liang, S., Tsutakawa, S. E., Lees-Miller, S. P., Nadal, M., Tainer, J. A., Blundell, T. L., Strick, T. R. 2018; 25 (6): 482-487

  Abstract

  Repairing DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nanomanipulation, allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and the addition of PAXX extends this lifetime to ~2 s. Further addition of XRCC4, XLF and ligase IV results in minute-scale synapsis and leads to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kilocalories per mole. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity.

  View details for DOI 10.1038/s41594-018-0065-1

  View details for PubMedID 29786079

  View details for PubMedCentralID PMC5990469

• The histone H3.3K36M mutation reprograms the epigenome of chondroblastomas. Science (New York, N.Y.) Fang, D., Gan, H., Lee, J. H., Han, J., Wang, Z., Riester, S. M., Jin, L., Chen, J., Zhou, H., Wang, J., Zhang, H., Yang, N., Bradley, E. W., Ho, T. H., Rubin, B. P., Bridge, J. A., Thibodeau, S. N., Ordog, T., Chen, Y., van Wijnen, A. J., Oliveira, A. M., Xu, R. M., Westendorf, J. J., Zhang, Z. 2016; 352 (6291): 1344-8

  Abstract

  More than 90% of chondroblastomas contain a heterozygous mutation replacing lysine-36 with methionine-36 (K36M) in the histone H3 variant H3.3. Here we show that H3K36 methylation is reduced globally in human chondroblastomas and in chondrocytes harboring the same genetic mutation, due to inhibition of at least two H3K36 methyltransferases, MMSET and SETD2, by the H3.3K36M mutant proteins. Genes with altered expression as well as H3K36 di- and trimethylation in H3.3K36M cells are enriched in cancer pathways. In addition, H3.3K36M chondrocytes exhibit several hallmarks of cancer cells, including increased ability to form colonies, resistance to apoptosis, and defects in differentiation. Thus, H3.3K36M proteins reprogram the H3K36 methylation landscape and contribute to tumorigenesis, in part through altering the expression of cancer-associated genes.

  View details for DOI 10.1126/science.aae0065

  View details for PubMedID 27229140

  View details for PubMedCentralID PMC5460624