CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

Structural Basis for a Polθ Helicase Small-Molecule Inhibitor Revealed by Cryo-EM

DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC50 against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers

Changes in chromatin structure during processing of wax-embedded tissue sections

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ

VIBRATIONAL CIRCULAR DICHROISM OF TARTARIC ACID ESTERS. CONFIGURATIONAL CORRELATION AND UNIQUE SENSITIVITY TO HYDROGEN BONDING EFFECTS

$^{1}$ P. L. Polavarapu, '40th Symposium on Molecular Spectroscopy,' Columbus, OH (1985). Paper WE3 Address of Chandramouly, Ewig and Polavarapu: Department of Chemistry, Vanderbilt University, Nashville, TN 37235Author Institution:Vibrational circular dichroism (VCD) in tartaric acid, dimethyl tartrate, diethyl tartrate and diisopropyl tartrate have been measured in $CCl_{4}$ and DMSO solvents. These measurements, which arc in addition to those reported $earlier,^{1}$ inidicate that the VCD associated with $C^{\ast}-O$ stretching vibrations is identical in all the molecules of the present series. However, VCD associated with the $C=O$ stretching vibration differs from molecule to molecule. These differences are attributed to the well known internal hydrogen bonding and the influence there on from the substituents in COOR group. In order to get a physical picture of the different possible hydrogen bonded conformers, ab initio calculations have been carried out and the minimum energy conformers are identified. The conclusions emerging from these studies point to the extreme sensitivity of VCD, associated with the $C=O$ stretching vibrations, to the nature of internal hydrogen bonding

Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium.

Cell-cell communication is essential for tissue homeostasis, but its contribution to disease prevention remains to be understood. We demonstrate the involvement of connexin 43 (Cx43) and related gap junction in epithelial homeostasis illustrated by polarity-mediated cell cycle entry and mitotic spindle orientation (MSO). Cx43 is restricted to the apicolateral membrane of phenotypically normal breast luminal epithelial cells in 3D culture and in vivo. Chemically-induced blockade of gap junction intercellular communication (GJIC) as well as the absence of Cx43 disrupt the apicolateral distribution of polarity determinant, tight junction marker ZO-1 and lead to random MSO and cell multilayering. Induced expression of Cx43 in cells that normally lack this protein reestablishes polarity and proper MSO in 3D culture. The Cx43-directed MSO implicates PI3K-aPKC signaling, and Cx43 coprecipitates with signaling node proteins β-catenin and ZO-2 in the polarized epithelium. The distribution of Cx43 is altered by pro-inflammatory breast cancer risk factors like leptin and high fat diet, as shown in cell culture and on tissue biopsy sections. The control of polarity-mediated quiescence and MSO may contribute to the tumor suppressive role of Cx43.</jats:p

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ