Browsing by Author "Demeler, Borries"

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    Characterization of the Interaction between the Cohesin Subunits Rad21 and SA1/2
    (Public Library of Science, 2013) Zhang, Nenggang; Jiang, Yunyun; Mao, Qilong; Demeler, Borries; Tao, Yizhi Jane; Pati, Debananda
    The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits, namely Rad21/Mcd1/Scc1, Smc1, Smc3, and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is generated during DNA replication and maintained until the onset of anaphase. Among the many proposed models of the cohesin complex, the ﾒcoreﾒ cohesin subunits Smc1, Smc3, and Rad21 are almost universally displayed as tripartite ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core subunit SA1/SA2. To gain deeper insight into the function of SA1/SA2 in the cohesin complex, we have mapped the interactive regions of SA2 and Rad21 in vitro and ex vivo. Whereas SA2 interacts with Rad21 through a broad region (301ﾖ750 aa), Rad21 binds to SA proteins through two SA-binding motifs on Rad21, namely N-terminal (NT) and middle part (MP) SA-binding motif, located At 60-81 aa of the N-terminus and 383ﾖ392 aa of the MP of Rad21, respectively. The MP SA-binding motif is a 10 amino acid, a-helical motif. Deletion of these 10 amino acids or mutation of three conserved amino acids (L385, F389, and T390) in this ahelical motif significantly hinders Rad21 from physically interacting with SA1/2. Besides the MP SA-binding motif, the NT SAbinding motif is also important for SA1/2 interaction. Although mutations on both SA-binding motifs disrupt Rad21-SA1/2 interaction, they had no apparent effect on the Smc1-Smc3-Rad21 interaction. However, the Rad21-Rad21 dimerization was reduced by the mutations, indicating potential involvement of the two SA-binding motifs in the formation of the two-ring handcuff for chromosomal cohesion. Furthermore, mutant Rad21 proteins failed to significantly rescue precocious chromosome separation caused by depletion of endogenous Rad21 in mitotic cells, further indicating the physiological significance of the two SA-binding motifs of Rad21.
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    DNA supercoiling-induced shapes alter minicircle hydrodynamic properties
    (Oxford University Press, 2023) Waszkiewicz, Radost; Ranasinghe, Maduni; Fogg, Jonathan M; Catanese, Daniel J, Jr.; Ekiel-Jeżewska, Maria L; Lisicki, Maciej; Demeler, Borries; Zechiedrich, Lynn; Szymczak, Piotr
    DNA in cells is organized in negatively supercoiled loops. The resulting torsional and bending strain allows DNA to adopt a surprisingly wide variety of 3-D shapes. This interplay between negative supercoiling, looping, and shape influences how DNA is stored, replicated, transcribed, repaired, and likely every other aspect of DNA activity. To understand the consequences of negative supercoiling and curvature on the hydrodynamic properties of DNA, we submitted 336 bp and 672 bp DNA minicircles to analytical ultracentrifugation (AUC). We found that the diffusion coefficient, sedimentation coefficient, and the DNA hydrodynamic radius strongly depended on circularity, loop length, and degree of negative supercoiling. Because AUC cannot ascertain shape beyond degree of non-globularity, we applied linear elasticity theory to predict DNA shapes, and combined these with hydrodynamic calculations to interpret the AUC data, with reasonable agreement between theory and experiment. These complementary approaches, together with earlier electron cryotomography data, provide a framework for understanding and predicting the effects of supercoiling on the shape and hydrodynamic properties of DNA.
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    Hydroxyproline-free Single Composition ABC Collagen Heterotrimer
    (American Chemical Society, 2013) Jalan, Abhishek A.; Demeler, Borries; Hartgerink, Jeffrey D.
    Hydroxyproline plays a major role in stabilizing collagenous domains in eukaryotic organisms. Lack of this modification is associated with significant lowering in thermal stability of the collagen triple helix and may also affect fibrillogenesis and folding of the peptide chains. In contrast, even though bacterial collagens lack hydroxyproline, their thermal stability is comparable to fibrillar collagen. This has been attributed to the high frequency of charged amino acids found in bacterial collagen. Here we report a thermally stable hydroxyproline-free ABC heterotrimeric collagen mimetic system composed of decapositive and decanegative peptides and a zwitterionic peptide. None of the peptides contain hydroxyproline and furthermore the zwitterionic peptide does not even contain proline. The heterotrimer is electrostatically stabilized via multiple interpeptide lysine-aspartate and lysine-glutamate salt-bridges and maintains good thermal stability with a melting temperature of 37 °C. The ternary peptide mixture also populates a single composition ABC heterotrimer as confirmed by circular dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy. This system illustrates the power of axial salt-bridges to direct and stabilize the self-assembly of a triple helix and may be useful in analogous designs in expression systems where the incorporation of hydroxyproline is challenging.