Despite extensive study, there is little experimental information available as to which of the deoxyribose hydrogen atoms of duplex DNA reacts most with the hydroxyl radical. To investigate this question, we prepared a set of double-stranded DNA molecules in which deuterium had been incorporated specifically at each position in the deoxyribose of one of the four nucleotides. We then measured deuterium kinetic isotope effects on the rate of cleavage of DNA by the hydroxyl radical. These experiments demonstrate that the hydroxyl radical reacts with the various hydrogen atoms of the deoxyribose in the order 5 H > 4 H > 3 H Ϸ 2 H Ϸ 1 H. This order of reactivity parallels the exposure to solvent of the deoxyribose hydrogens. Our work therefore reveals the structural basis of the reaction of the hydroxyl radical with DNA. These results also provide information on the mechanism of DNA damage caused by ionizing radiation as well as atomic-level detail for the interpretation of hydroxyl radical footprints of DNA-protein complexes and chemical probe experiments on the structure of RNA and DNA in solution.The hydroxyl radical (⅐OH), the quintessential reactive oxygen species, is the mediator of much of the DNA damage caused by ionizing radiation (1). This damage includes strand breaks, which are initiated by abstraction of a deoxyribose hydrogen atom by the hydroxyl radical. DNA strand breaks induced by the hydroxyl radical also form the basis of a widely used method for making footprints of DNA-protein complexes (2, 3) and for studying the structure of DNA (4) and RNA (5) in solution. The key experimental advantage of the hydroxyl radical as a chemical probe is that it effects DNA cleavage with no base-or sequence-specificity (6-8). The hydroxyl radical produces highly detailed footprints that yield information about DNA structure (4, 7) and protein-DNA interactions (3, 8, 9) at single-nucleotide resolution.Mechanistic information on the reaction of the hydroxyl radical with nucleic acids will benefit our understanding of radiation damage to DNA as well as the interpretation of chemical probe experiments. The extensive literature on the radiation chemistry of DNA (1) is a rich source of mechanistic possibilities. Not surprisingly, because of the high reactivity of the hydroxyl radical, a wide spectrum of products has been detected on treatment of the constituents of DNA (nucleic bases, nucleosides, nucleotides, or simple-sequence singlestranded DNA, for example) with ionizing radiation (1). It has been more difficult to conduct similarly detailed experiments on the biologically relevant duplex form of DNA. It is not hard to conceive, though, that the hydroxyl radical might react in a different manner with double-stranded DNA compared with simpler nucleic acid systems because the shape of the double helix would strongly influence the accessibility of the various COH bonds in DNA.Until now, the extent of cleavage at a particular nucleotide in a hydroxyl radical footprinting experiment only could be interpreted at th...

We have shown previously that UV radiation and other DNA-damaging agents induce the ubiquitination of a portion of the RNA polymerase II large subunit (Pol II LS). In the present study UV irradiation of repaircompetent fibroblasts induced a transient reduction of the Pol II LS level; new protein synthesis restored Pol II LS to the base-line level within 16 -24 h. In repair-deficient xeroderma pigmentosum cells, UV radiation-induced ubiquitination of Pol II LS was followed by a sustained reduction of Pol II LS level. In both normal and xeroderma pigmentosum cells, the ubiquitinated Pol II LS had a hyperphosphorylated COOH-terminal domain (CTD), which is characteristic of elongating Pol II. The portion of Pol II LS whose steady-state level diminished most quickly had a relatively hypophosphorylated CTD. The ubiquitinated residues did not map to the CTD. Importantly, UV-induced reduction of Pol II LS level in repair-competent or -deficient cells was inhibited by the proteasome inhibitors lactacystin or MG132. These data demonstrate that UV-induced ubiquitination of Pol II LS is followed by its degradation in the proteasome. These results suggest, contrary to a current model of transcription-coupled DNA repair, that elongating Pol II complexes which arrest at intragenic DNA lesions may be aborted rather than resuming elongation after repair takes place.

The COVID pandemic forced many higher education institutions to pivot and switch to an online learning environment with minimal preparation. This transition was put in place during the middle of the spring semester, in March. During this transition, instructors had to quickly learn the tools of online teaching, navigate platforms like Webex and Zoom, and adapt their lectures to an online format. One of the biggest challenges during this transition was to administer common online exams to high enrollment undergraduate classes, such as general chemistry and organic chemistry, without compromising the integrity of the exam. The in-person chemistry common exam that is normally administered to students at New Jersey Institute of Technology (NJIT) had both multiple-choice and open-ended questions. In order to replicate this online, and to reduce the potential temptation to cheat, a robust multiple-choice and open-ended exam with multiple versions was required. Furthermore, this online exam had to be easily administered through our learning management system. What was developed included a blueprint for an online exam intended for large-scale distribution over multiple days with safeguards in place to protect the integrity of the examination. The exam employed deferred grading, a lockdown browser, multiple question variants, time controls, and controlled access to the completed exam to combat potential cheating. The exam resulted in average scores that were comparable to in-person exam scores from previous semesters, validating the proposed approach. In addition, polling results after administration of the exam showed strong student satisfaction with exam design and directions and student preference for webcam proctored exams.

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The human Betacoronavirus SARS-CoV-2 is a novel pathogen claiming millions of lives and causing a global pandemic that has disrupted international healthcare systems, economies, and communities. The virus is fast mutating and presenting more infectious but less lethal versions. Currently, some small-molecule therapeutics have received FDA emergency use authorization for the treatment of COVID-19, including Lagevrio (molnupiravir) and Paxlovid (nirmaltrevir/ritonavir), which target the RNA-dependent RNA polymerase and the 3CLpro main protease, respectively. Proteins downstream in the viral replication process, specifically the nonstructural proteins (Nsps1−16), are potential drug targets due to their crucial functions. Of these Nsps, Nsp4 is a particularly promising drug target due to its involvement in the SARS-CoV viral replication and double-membrane vesicle formation (mediated via interaction with Nsp3). Given the degree of sequence conservation of these two Nsps across the Betacoronavirus clade, their protein−protein interactions and functions are likely to be conserved as well in SARS-CoV-2. Through AlphaFold2 and its recent advancements, protein structures were generated of Nsp3 and 4 lumenal loops of interest. Then, using a combination of molecular docking suites and an existing library of lead-like compounds, we virtually screened 7 million ligands to identify five putative ligand inhibitors of Nsp4, which could present an alternative pharmaceutical approach against SARS-CoV-2. These ligands exhibit promising lead-like properties (ideal molecular weight and log P profiles), maintain fixed-Nsp4-ligand complexes in molecular dynamics (MD) simulations, and tightly associate with Nsp4 via hydrophobic interactions. Additionally, alternative peptide inhibitors based on Nsp3 were designed and shown in MD simulations to provide a highly stable binding to the Nsp4 protein. Finally, these therapeutics were attached to dendrimer structures to promote their multivalent binding with Nsp4, especially its large flexible luminal loop (Nsp4LLL). The therapeutics tested in this study represent many different approaches for targeting large flexible protein structures, especially those localized to the ER. This study is the first work targeting the membrane rearrangement system of viruses and will serve as a potential avenue for treating viruses with similar replicative function.