Maha Zewail-Foote

A sequence-specific threading tetra-intercalator with an extremely slow dissociation rate constant

A long-lived and sequence specific ligand-DNA complex would make possible the modulation of biological processes for extended periods. We have been investigating the threading polyintercalation approach to DNA recognition in which chains of aromatic units thread back and forth repeatedly through the double helix. Here we report the preliminary sequence specificity and detailed kinetic analysis of a structurally characterized threading tetraintercalator. Specific binding on a relatively long DNA strand was observed, strongly favoring a predicted 14-base pair sequence. Kinetic studies revealed a multi-step association process and specificity was found to derive primarily from large differences in dissociation rates. Importantly, the rate-limiting dissociation rate constant of the tetraintercalator complex dissociating from its preferred binding site was extremely slow, corresponding to a 16 day half-life, making it one of the longer non-covalent complex half-lives yet measured, and, to the best of our knowledge, the longest for a DNA binding molecule.

The antitumor agent ecteinascidin 743: characterization of its covalent DNA adducts and chemical stability.

Ecteinascidin 743 (Et 743), a natural product derived from the Caribbean tunicate Eteinascidia turbinata, is a potent antitumor agent currently in phase II clinical trials. Et 743 binds in the minor groove of DNA, forming covalent adducts by reacting with N2 of guanine. Although DNA is considered to be the macromolecular receptor for Et 743, the precise mechanism by which Et 743 exerts its remarkable antitumor activity has not yet been elucidated. The aim of this study is to provide a rationale for the antitumor activity of Et 743 by studying its fundamental interactions with DNA at the molecular level. First, DNA structural distortions induced by Et 743 were characterized using gel electrophoresis. Surprisingly, Et 743 bends DNA toward the major groove, a unique feature among DNA-interactive agents that occupy the minor groove. Second, in order to gain further insight into the molecular basis behind the apparent sequence selectivity of Et 743, the stability and structure of Et 743 adducts at different target sequences were determined. On the basis of this data, the overall stability of the Et 743-DNA adducts was found to be governed by the DNA target sequence, where the inability of Et 743 to form optimum bonding networks with its optimum recognition sites leads to the formation of an unstable adduct. Consequently, the reaction of Et 743 with DNA is reversible, and the rate of the reverse reaction is a function of the target and flanking sequences. The results from this study demonstrate that Et 743 differs from other DNA alkylating agents by its effects on DNA structure and sequence-dependent chemical stability. This information provides important insight into the underlying mechanisms for its unique profile of antitumor activity.

Reproductive success and mate choice in Long-Evans rats.

In the present study, mating behavior was observed in female rats that were given the opportunity to mate with two male rats simultaneously. Eleven sexually naïve, naturally cycling rats were assigned to one pair of sexually experienced cohabitating male rats. Each female rat was mated during proestrous with her assigned pair of males. Eight females successfully became pregnant and gave birth to healthy pups (approximately 9 pups/litter). After the offspring were weaned, paternity of each pup was determined using microsatellite DNA markers. Based on this analysis, it was determined that one male in each pair sired the majority of the pups in the litter. No male sexual behaviors or characteristics distinguished male rats that sired the majority of pups from those that sired few or none of the pups. Furthermore, neither female mate preference nor measures of paced mating behavior differed between male rats that had a reproductive advantage from those that did not. Finally, ejaculation order also failed to predict reproductive success of the male rats. Together, these results suggest that reproductive success of some male rats may not depend on specific mating behaviors, but rather their success may depend on physiological or genetic traits that make them unique.

Sexual Attractiveness in Male Rats Is Associated with Greater Concentration of Major Urinary Proteins

ABSTRACT Female rats show a distinct attraction for males. This attraction remains consistent without the necessity for the physical presence of the male. However, the identity of the olfactory cues contributing to attraction in rats remains unknown. Rat urine contains copious amounts of major urinary proteins (MUPs). Here, we investigated the hypothesis that MUPs mediate sexual attractiveness in rats. We first demonstrated that a member of a male dyad receiving greater copulatory opportunities in competitive mate choice tests excrete greater amounts of MUPs. Furthermore, the amount of male MUPs positively correlated with both copulatory opportunities received and female exploration of the urine. Using females and a two-choice olfactory attraction test, we demonstrated that urinary fractions containing MUPs were sufficient to induce attraction and that male MUPs activated neurons in the posterodorsal medial amygdala in female rats. Taken together, these results suggest that olfactory cues associated with MUPs act as an attractant to female rats in estrus.

Methamphetamine induces DNA damage in specific regions of the female rat brain.

Methamphetamine (METH) is a highly addictive psychostimulant that has been shown to produce neurotoxicity. Methamphetamine increases the release of dopamine by reversing the direction of monoamine transporter proteins, leading to the formation of reactive oxygen species in the brain. In this study, we examined the effect of METH on DNA damage in vivo using the single cell gel electrophoresis assay (comet assay) under two different conditions. Rats treated with multiple doses of METH (10 mg/kg × 4) showed significant levels of DNA damage in the nucleus accumbens and striatum, both dopamine‐rich areas. In contrast, a single dose of METH did not lead to significant levels of DNA damage in any of the dopamine‐rich brain regions that were tested. Overall, the results of our study demonstrate that METH produces greater oxidative DNA damage in brain areas that receive greater dopamine innervation.

Alternative DNA structure formation in the mutagenic human c-MYC promoter

Abstract Mutation ‘hotspot’ regions in the genome are susceptible to genetic instability, implicating them in diseases. These hotspots are not random and often co-localize with DNA sequences potentially capable of adopting alternative DNA structures (non-B DNA, e.g. H-DNA and G4-DNA), which have been identified as endogenous sources of genomic instability. There are regions that contain overlapping sequences that may form more than one non-B DNA structure. The extent to which one structure impacts the formation/stability of another, within the sequence, is not fully understood. To address this issue, we investigated the folding preferences of oligonucleotides from a chromosomal breakpoint hotspot in the human c-MYC oncogene containing both potential G4-forming and H-DNA-forming elements. We characterized the structures formed in the presence of G4-DNA-stabilizing K+ ions or H-DNA-stabilizing Mg2+ ions using multiple techniques. We found that under conditions favorable for H-DNA formation, a stable intramolecular triplex DNA structure predominated; whereas, under K+-rich, G4-DNA-forming conditions, a plurality of unfolded and folded species were present. Thus, within a limited region containing sequences with the potential to adopt multiple structures, only one structure predominates under a given condition. The predominance of H-DNA implicates this structure in the instability associated with the human c-MYC oncogene.

Kin discrimination in prepubescent and adult Long–Evans rats

The present study investigated the preference of prepubescent and adult rats for an unrelated conspecific over a closely related conspecific (e.g., father, mother). Preference was measured by the amount of time spent in the vicinity of the stimulus animals as well as who was visited first. To prevent mating behavior, stimulus animals were housed behind wire-mesh. Experiment 1 determined if adult female offspring prefer an unrelated, unfamiliar adult male or their father. The preference of adult female rats was independent of kinship. Experiment 2 evaluated the preference of prepubescent female and male offspring for an unrelated, unfamiliar adult male or their father. The preference of prepubescent female and male rats was also independent of kinship. Experiment 3 evaluated the preference of adult male offspring for an unrelated, unfamiliar adult female or their mother. The preference of adult male rats was independent of kinship. In summary, prepubescent and adult rats do not demonstrate preference for kin vs. non-kin (as measured by time spent near stimulus animals or who was visited first). Although kin recognition provides a mechanism for inbreeding avoidance (Wilson, 1987), in the present study adult rats show no evidence of inbreeding avoidance.