Browsing by Author "Lu, Xiaotong"

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    High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL
    (Elsevier, 2023) Kaur, Parminder; Lu, Xiaotong; Xu, Qi; Irvin, Elizabeth Marie; Pappas, Colette; Zhang, Hongshan; Finkelstein, Ilya J.; Shi, Zhubing; Tao, Yizhi Jane; Yu, Hongtao; Wang, Hong
    3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.
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    Structural and Biochemical Characterization of the Influenza D Virus Matrix Protein Reveals its Mechanisms for Membrane and vRNP Association
    (2023-04-21) Lu, Xiaotong; Tao, Yizhi Jane; Shamoo, Yousif
    First discovered in 2012, Influenza D virus primarily infects livestock such as bovine and swine. Influenza D virus has a single-stranded, negative-sense RNA genome with seven gene segments encoding a total of nine proteins. The M segment, or segment 6, expresses the matrix protein DM1 and the precursor protein DM, which is further processed to generate the ion channel DM2. DM1 is involved in virus budding and also serves as a bridge between the viral membrane and viral ribonuclear protein complex (vRNP) to facilitate genome packaging. In this project, I first used peptide mass fingerprinting to confirm the protein sequence of DM1 from infectious viral particles. The N-terminal domain of DM1 (DM1-N) containing the first 169 amino acids were crystallized and its structure determined to 2.3-Å resolution. The structure of DM1-N contains nine -helices and is highly homologous to that of the influenza A virus M1. Two positively charged surfaces formed by helices 5 and 6 are likely to be involved in membrane and vRNP interactions. Through liposome flotation assays, it was found that DM1 binds to membrane with DM1-N, but efficient membrane association requires inter-molecular oligomerization of DM1. Our structural modeling indicated that DM1 oligomerization involves an intricate interaction network involving DM1N from one subunit interacting with DM1C from the adjacent molecule. DM1-L, a mutant with an elongated inter-domain linker designed to disrupt intermolecular interaction and prevent oligomerization, had a much weakened membrane binding ability compared to wild-type DM1, consistent with our hypothesis. Fluorescence anisotropy assays demonstrated that DM1 exhibited a high binding affinity for single-stranded RNA, but neither DM1-N nor DM1-C alone was sufficient for this activity. Molecular dynamic simulation revealed that RNA consistently bound to the large cleft formed between DM1-N and DM1-C, thus providing a novel model that can be used to explain the role of DM1 in genome packaging. The interaction between DM1 and NP was also characterized using several techniques including MST, OCTET, and dot blot assays. My results showed that both DM1-N and DM1-C contributes to NP binding, but the interaction is primarily mediated by DM1-N. Overall, this project provides valuable insights into the biological functions of DM1 and its role in virus assembly. My results also suggest promising directions that DM1 can be further exploited for future antiviral drug development.