DNA double-strand breaks, one of the most cytotoxic forms of DNA damage, can be detected and repaired by the fast-responding and tightly regulated non-homologous end-joining (NHEJ) machinery. NHEJ factors are targets for the development of cancer therapeutics and are essential for the generation of antibody and antigen receptor diversity in immune cells. Core NHEJ factors (Ku70/80 heterodimer (Ku), catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF) form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs free, short-range (SR) state to align and repair the DSB ends. Using single-particle Cryo-Election Microscopy (Cryo-EM), we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Integrated modeling with both experimental reconstruction and in silico structural prediction reveals how an accessory NHEJ scaffolding factor, PAXX, stabilizes the LR complex during ATP-dependent DNA-PKcs signaling. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. In addition, we captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ.
12:30-1:00 Troponin Enhanceropathies: A novel role for the troponin genes
Jenna Thuma,
Davis lab
(with Yvette Wang, Madhoolika Bisht, Svetlana Tikunova, and Jonathan Davis, Department of Physiology and Cell Biology)
Striated muscle contraction is controlled at the level of the thin filament by the troponin complex. This complex consists of three proteins: the calcium binding subunit, troponin C (TnC), the inhibitory subunit, troponin I (TnI), and the tropomyosin binding subunit, troponin T (TnT). These can come in different isoforms depending on the type of striated muscle, fast or slow skeletal and cardiac. Cardiac troponin's protein role has already been well known and documented, in both health and disease; however, we propose a novel role for all three cardiac troponins at the DNA level. Only about 2% of the genome codes for proteins, the rest of the genome falls into one of several other categories such as repeat sequences, pseudogenes, or regulatory elements. Regulatory elements are important genomic elements that control the expression of up or downstream protein coding genes by enabling cell specific and time specific expression. These types of elements are generally found intergenically or within the introns of protein coding genes, however, rarely, they can be found within exons (~5-10%). Shockingly, we found evidence that all three cardiac troponin genes contain exonic regulatory elements. As a consequence of this, any nucleotide mutation in their exonic regulatory element could not only change protein structure and function, but also the function of the regulatory element resulting in unpredicted effects. For example, the regulatory element within cardiac troponin C is predicted to regulate several genes important for early development and, consistent with this, when we mutate this region we lose all homozygous embryos in both mice and rats. Additionally, the regulatory element within cardiac troponin I appears to interact with two genes, slow skeletal TnT and Dnaaf3. cTnI knockout mice have already been shown to have skeletal muscle (diaphragm) weakness by JP Jin's group and we are documenting several thoracic and abdominal organ abnormalities consistent with an issue in Dnaaf3. Thus, we are proposing a novel DNA regulatory role for the cardiac troponins, independent of their protein function, that, when mutated, may cause non-cardiac disease.
Ribosomal RNAs are transcribed as part of larger precursor molecules. In Escherichia coli, complementary RNA segments flank each rRNA and form long leader-trailer (LT) helices, which are crucial for subunit biogenesis in the cell. A previous study of 15 representative species suggested that most but not all prokaryotes contain LT helices. Here, we use a combination of in silico folding and covariation methods to identify and characterize LT helices in 4464 bacterial and 260 archaeal organisms. Our results suggest that LT helices are present in all phyla, including Deinococcota, which had previously been suspected to lack LT helices. In very few organisms, our pipeline failed to detect LT helices for both 16S and 23S rRNA. However, a closer case-by-case look revealed that LT helices are indeed present but escaped initial detection. Over 3600 secondary structure models, many well supported by nucleotide covariation, were generated. These structures show a high degree of diversity. Yet, all exhibit extensive base-pairing between the leader and trailer strands, in line with a common and essential function.
Last update: 9/23/2024, Ralf Bundschuh