Alexander M. Spring

Recognition of Damaged DNA: Structure and Dynamic Markers

DNA damage, a consequence of external factors and inherent metabolic processes, is omnipresent. Nature has devised multiple strategies to safeguard the genetic information and developed intricate repair mechanisms and pathways to reverse an array of different DNA lesions, including mismatches. Failure of the DNA repair systems may result in mutation, premature ageing, and cancer. In this review, we focus on structural and dynamic aspects of detection of lesions in base excision and mismatch repair. A thorough understanding of repair, pathways, and regulation is necessary to develop strategies for targeting DNA‐related pathologies.  © 2010 Wiley Periodicals, Inc. Med Res Rev, 32, No. 3, 659–683, 2012

Structural basis of the RNase H1 activity on stereo regular borano phosphonate DNA/RNA hybrids.

Numerous DNA chemistries for improving oligodeoxynucleotide (ODN)-based RNA targeting have been explored. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODNs are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH(3) modification on the nucleic acid structure and RNase H1 enzyme activity, we have investigated two DNA/RNA hybrids containing either a R(P) or S(P) BH(3) modification by nuclear magnetic resonance (NMR) spectroscopy. T(M) studies show that the stabilities of R(P) and S(P) modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to that of an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. To characterize such similar structures, we used a large number of NMR restraints (including dipolar couplings and backbone torsion angles) to determine structural features that were important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total R(x) values of <6%) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In the case of the S(P) modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the S(P) BH(3) group in the major groove while the R(P) BH(3) group points away from the DNA. However, this creates a potential clash of the R(P) BH(3) groups with important RNase H1 residues in a complex, while the S(P) BH(3) groups could be tolerated. We therefore predict that on the basis of our NMR structures a fully R(P) BH(3) DNA/RNA hybrid would not be a substrate for RNase H1.

Thermodynamic Profiling of HIV RREIIB RNA-Zinc Finger Interactions

The interactions between the HIV Rev-responsive element (RRE) RNA and the HIV regulatory protein Rev, are crucial for the HIV life-cycle. Earlier, we showed that single C(2)H(2) zinc fingers (znfs) have the same binding site as the Rev peptide and exhibit nanomolar affinity. In this study, the specific role of amino acid side chains and molecular processes involved with complex formation were investigated by perturbation of the binding energetics via changes in temperature, pH, buffers, and salt concentrations, as well as znf and RNA mutations, by isothermal titration calorimetry. Interestingly, despite the large cationic charge on the znfs, the number of interactions with the RNA phosphate backbone was lower than intuitively expected. The presence of binding induced protonation was established by ITC and localized by NMR to a histidine on the znf beta-sheet. The DeltaC(p) of znf-RNA binding was observed to be substantially negative and could not be accounted for by conventional solvent-accessible surface area models. An alternative model, based on the extent of hydrogen bond changes as a result of differences in ligand-induced water displacement at the binding site, provided reasonable explanation of the trends in DeltaC(p), as well as DeltaH and DeltaS. Our studies show that incorporation of favorable interactions at the solvent-excluded binding interface can be used to alleviate the unfavorable enthalpic penalties of displacing water molecules from the hydrated RNA surface.

Combination Treatment with Sublethal Ionizing Radiation and the Proteasome Inhibitor, Bortezomib, Enhances Death-Receptor Mediated Apoptosis and Anti-Tumor Immune Attack

Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity.

The importance of fitting in: conformational preference of selenium 2′ modifications in nucleosides and helical structures

Selenomethionine incorporation has proven useful in X-ray crystallography of proteins to obtain phase information. In nucleic acids, the introduction of selenium to different positions is beneficial for solving the phase problem as well, but its addition to the 2′ position also significantly enhances the crystal formation. The selenium modification in a single nucleotide shows a preference towards 2′-endo sugar puckering, which is in conflict with existing crystal structures where the duplex incorporated 2′-selenium-modified nucleotide is exclusively found in a 3′-endo conformation. Our work provides a rationale why 2′-selenium modifications facilitate crystallization despite this contradictory behavior.

Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes

Accumulating evidence shows the 26S proteasome is involved in the regulation of gene expression. We and others have demonstrated that proteasome components bind to sites of gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear, although prior observations suggest both proteolytic and non-proteolytic activities. Here, we define novel, non-proteolytic, roles for each of the three 19S heterodimers, represented by the 19S ATPases Sug1, S7, and S6a, in mammalian gene expression using the inflammatory gene CIITApIV. These 19S ATPases are recruited to induced CIITApIV promoters and also associate with CIITA coding regions. Additionally, these ATPases interact with elongation factor PTEFb complex members CDK9 and Hexim-1 and with Ser5 phosphorylated RNA Pol II. Both the generation of transcripts from CIITApIV and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by siRNA mediated knockdown of these 19S ATPases. Together, these results define novel roles for 19S ATPases in mammalian gene expression and indicate roles for these ATPases in promoting transcription processes.

Supercooled aqueous nuclear magnetic resonance using agarose gels

Low-temperature nuclear magnetic resonance (NMR), especially under supercooled conditions, can give critical insight into biomolecular systems via slowed dynamics and exchange rates. These conditions can also increase correlation times of small molecules, potentially allowing for NMR structural study of small molecules at moderate field strengths. Agarose gels allow for supercooled conditions and are simple to prepare, invisible to NMR, and noninteractive with most biomolecules and organics. Here we demonstrate their use with nucleic acids, small organic molecules, and peptides.

Facilitated Assignment of Adenine H2 Resonances in Oligonucleotides using Homonuclear Long-range Couplings

The small homonuclear coupling constant between H2 and H8 protons of adenine (0.5 +/- 0.2 Hz) was determined and exploited to assign the H2 proton of adenine residues in DNA oligonucleotides. The utility of this approach is demonstrated for two different DNA sequences in D(2)O, for which we show a rapid and unambiguous assignment of the A H2 resonances.