Nuclear extracts derived from HeLa and Drosophila nelanogaster Kc cell lines have been found to correct single base-base mispars within open circular DNA heteroduplexes containing a strand-specific, site-specific incision located 808 base pairs from the mismatch. Correction in both extract systems is strand specific, being highly biased to the incised DNA strand. Different mispairs within a homologous set of heteroduplexes were processed with different efficiencies (G-T > GG ARC > CC), and correction was accompanied by mismatch-dependent DNA synthesis localized to the region snning the mispair and the strand break, thus demonstrating that mismatch recognition is associated with the repair reaction. Correction ofeach of these heteroduplexes was abolished by aphidicolin but was relatively insensitive to the presence of high concentrations of ddTTP, indicating probable involvement of a and/or 8 class DNA polymerase(s). These findings suggest that higher eukaryotic cells possess a general, strand-specific mismatch repair system analogous to the Eseherichia coli mutHLS and the Streptococcus pneumoniae hexAB pathways, systems that contribute in a major way to the genetic stability of these bacterial species.DNA mismatch correction is best understood in Escherichia coli, where the process has been addressed by both biological and biochemical methods. This organism possesses several systems for mismatch correction, with the most extensively studied being the MutHLS-dependent, methyl-directed pathway that serves to enhance the accuracy of chromosome replication and ensures the fidelity ofgenetic exchange (1-4). This system, which is capable of processing a variety of mispairs, catalyzes a strand-specific reaction in which repair is targeted to a DNA strand that lacks d(GATC) methylation or contains a persistent strand break (5,6). Much less is known about the mechanisms and functions of mismatch repair in higher cells. Mutations in yeast PMSI, PMS2, and PMS3 loci confer a mutator phenotype, a high frequency of postmeiotic segregation, and a defect in mismatch correction (7-9). Although the mispair specificity of the PMS-dependent pathway is similar to that of the E. coli methyl-directed system and while the PMSJ gene product has been shown to be homologous to the bacterial MutL protein (9), no evidence for strand specificity of the yeast pathway has been presented. Compelling support for the occurrence of mismatch correction in several higher eukaryotic systems has also been reported. Evidence to this effect has been based on the demonstration that different mispairs are subject to differential processing when introduced into mammalian cells by heteroduplex transfection (10). As in the case of yeast, however, it is not clear whether higher eukaryotes possess a strand-specific correction pathway that is capable of processing a set of distinct mispairs in a manner analogous to the bacterial methyl-directed system. Although the transfection results of Hare and Taylor (11) have suggested that DNA strand breaks or met...

Acquired epilepsy (i.e., after an insult to the brain) is often considered to be a progressive disorder, and the nature of this hypothetical progression remains controversial. Antiepileptic drug treatment necessarily confounds analyses of progressive changes in human patients with acquired epilepsy. Here, we describe experiments testing the hypothesis that development of acquired epilepsy begins as a continuous process of increased seizure frequency (i.e., proportional to probability of a spontaneous seizure) that ultimately plateaus. Using nearly continuous surface cortical and bilateral hippocampal recordings with radiotelemetry and semiautomated seizure detection, the frequency of electrographically recorded seizures (both convulsive and nonconvulsive) was analyzed quantitatively for ϳ100 d after kainate-induced status epilepticus in adult rats. The frequency of spontaneous recurrent seizures was not a step function of time (as implied by the "latent period"); rather, seizure frequency increased as a sigmoid function of time. The distribution of interseizure intervals was nonrandom, suggesting that seizure clusters (i.e., short interseizure intervals) obscured the early stages of progression, and may have contributed to the increase in seizure frequency. These data suggest that (1) the latent period is the first of many long interseizure intervals and a poor measure of the time frame of epileptogenesis, (2) epileptogenesis is a continuous process that extends much beyond the first spontaneous recurrent seizure, (3) uneven seizure clustering contributes to the variability in occurrence of epileptic seizures, and (4) the window for antiepileptogenic therapies aimed at suppressing acquired epilepsy probably extends well past the first clinical seizure.

Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer's disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1,3-oxazino benzo-1,4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the "hinge" in the ligand-binding core "clamshell" that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.

An assay has been developed that permits analysis of DNA mismatch repair in cell-free extracts of Escherichia coli The method relies on repair of heteroduplex molecules of fl R229 DNA, which contain a base-pair mismatch within the single EcoRI site of the molecule. As observed with mismatch