Elizabeth R. Jamieson

Structural and Kinetic Studies of a Cisplatin-modified DNA Icosamer Binding to HMG1 Domain B

The high mobility group (HMG) domain is a DNA-binding motif found in the non-histone chromosomal proteins, HMG1 and HMG2, and some transcription factors. Experimental evidence has demonstrated that HMG-domain proteins can play a role in sensitizing cells to the anticancer drug cisplatin. Fluorescence resonance energy transfer (FRET) experiments were performed in the present study to investigate structural changes that accompany complex formation between the HMG domain B of HMG1 and a cisplatin-modified, 20-base pair double-stranded DNA probe containing fluorescein and rhodamine tethered at its two ends. The binding affinity of HMG1 domain B for the cisplatin-modified DNA probe was investigated in fluorescence titration experiments, and a value of 60 ± 30 nm was determined for the dissociation constant. Single photon counting methods were employed to measure the fluorescence lifetime of the fluorescein donor in the presence and absence of HMG1 domain B. These FRET experiments revealed a distance change that was used to estimate a bend angle of 80–95° for the cisplatin-modified DNA upon protein binding. Stopped-flow fluorescence spectroscopic experiments afforded kinetic parameters for HMG1 domain B binding to the cisplatin-modified DNA probe, with k on = 1.1 ± 0.1 × 109 m −1 s−1 andk off = 30 ± 4 s−1.

Identifying protein interactions with metal-modified DNA using microarray technology

Protein microarrays have been used extensively to identify protein–protein interactions; however, this technology has not been widely applied to protein–DNA interactions. In particular, this work demonstrates the utility of this technique for rapidly identifying interactions of proteins with metal-modified DNA. Protein macroarray experiments were carried out with high mobility group protein 1 (HMG-1) and cisplatin- and chromium-modified 50-mer oligonucleotides to demonstrate “proof of principle.” Commercially available protein microarrays containing many different classes of human proteins were then employed to search for additional interactions with cisplatin-modified DNA. The results of the microarray experiments confirmed some known interactions and, more importantly, identified many novel protein interactions, demonstrating the utility of this method as a rapid, high-throughput technique to discover proteins that interact with metal-modified DNA.

Effect of Base-Pairing Partner on the Thermodynamic Stability of the Diastereomeric Spiroiminodihydantoin Lesion

Oxidation of guanine by reactive oxygen species and high valent metals produces damaging DNA base lesions like 8-oxo-7,8-dihydroguanine (8-oxoG). 8-oxoG can be further oxidized to form the spiroiminodihydantoin (Sp) lesion, which is even more mutagenic. DNA polymerases preferentially incorporate purines opposite the Sp lesion, and DNA glycosylases excise the Sp lesion from the duplex, although the rate of repair is different for the two Sp diastereomers. To further understand the biological processing of the Sp lesion, differential scanning calorimetry studies were performed on a series of 15-mer DNA duplexes. The thermal and thermodynamic stabilities of each of the Sp diastereomers paired to the four standard DNA bases were investigated. It was found that, regardless of the base-pairing partner, the Sp lesion was always highly destabilizing in terms of DNA melting temperature, enthalpic stability, and overall duplex free energy. We found no significant differences between the two Sp diastereomers, but changing the base-pairing partner of the Sp lesion produced slight differences in stability. Specifically, duplexes with Sp:C pairings were always the most destabilized, whereas pairing the Sp lesion with a purine base modestly increased stability. Overall, these results suggest that, although the stability of the Sp diastereomers cannot explain the differences in the rates of repair by DNA glycosylases, the most stable base-pairing partners do correspond with the nucleotide preference of DNA polymerases.

Inorganic chemistry and IONiC: an online community bringing cutting-edge research into the classroom.

This Viewpoint highlights creative ways that members of the Interactive Online Network of Inorganic Chemists (IONiC) are using journal articles from Inorganic Chemistry to engage undergraduate students in the classroom. We provide information about specific educational materials and networking features available free of charge to the inorganic community on IONiCs web home, the Virtual Inorganic Pedagogical Electronic Resource (VIPEr, www.ionicviper.org ) and describe the benefits of joining this community.

Effect of the Spiroiminodihydantoin Lesion on Nucleosome Stability and Positioning.

DNA is constantly under attack by oxidants, generating a variety of potentially mutagenic covalently modified species, including oxidized guanine base products. One such product is spiroiminodihydantoin (Sp), a chiral, propeller-shaped lesion that strongly destabilizes the DNA helix in its vicinity. Despite its unusual shape and thermodynamic effect on double-stranded DNA structure, DNA duplexes containing the Sp lesion form stable nucleosomes upon being incubated with histone octamers. Indeed, among six different combinations of lesion location and stereochemistry, only two duplexes display a diminished ability to form nucleosomes, and these only by ∼25%; the other four are statistically indistinguishable from the control. Nonetheless, kinetic factors also play a role: when the histone proteins have less time during assembly of the core particle to sample both lesion-containing and normal DNA strands, they are more likely to bind the Sp lesion DNA than during slower assembly processes that better approximate thermodynamic equilibrium. Using DNase I footprinting and molecular modeling, we discovered that the Sp lesion causes only a small perturbation (±1-2 bp) on the translational position of the DNA within the nucleosome. Each diastereomeric pair of lesions has the same effect on nucleosome positioning, but lesions placed at different locations behave differently, illustrating that the location of the lesion and not its shape serves as the primary determinant of the most stable DNA orientation.