Many proteobacteria are able to monitor their population densities through the release of pheromones known as N-acylhomoserine lactones. At high population densities, these pheromones elicit diverse responses that include bioluminescence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis and symbiosis. Many of these regulatory systems require a pheromone-dependent transcription factor similar to the LuxR protein of Vibrio fischeri. Here we present the structure of a LuxR-type protein. TraR of Agrobacterium tumefaciens was solved at 1.66 A as a complex with the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) and its TraR DNA-binding site. The amino-terminal domain of TraR is an alpha/beta/alpha sandwich that binds OOHL, whereas the carboxy-terminal domain contains a helix turn helix DNA-binding motif. The TraR dimer displays a two-fold symmetry axis in each domain; however, these two axes of symmetry are at an approximately 90 degree angle, resulting in a pronounced overall asymmetry of the complex. The pheromone lies fully embedded within the protein with virtually no solvent contact, and makes numerous hydrophobic contacts with the protein as well as four hydrogen bonds: three direct and one water-mediated.

ContentsI. Introduction 939 A. Medicinal Motivation 941 B. Tools for Probing Ribozyme Mechanisms 943 C. Tools for Molecular Biology 943 II. The Chemistry of RNA Cleavage 943 A. Catalysts and Their Mechanisms 943 B. Assays for RNA Cleavage 944 1. Models for RNA (Both Good and Bad) 944 2. Dimeric Assays 946 3. Polymeric Assays 947 4. Chimeric Assays 948 III. Sequence-Specific Nucleophilic RNA Cleavage 949 A. Key Design Features 949 B. Preparation and Behavior of Sequence-Specific RNA Cleavage Agents 950 1. Hybrid Enzymes 950 2. Ribozyme Mimics Based on Metal Complexes 951 3. Ribozyme Mimics Based on Organic Catalysts 954 IV. Recent Ideas for the Design of Ribozyme Mimics 955 A. Creating RNA Bulges and Loops 955 B. Minimal Nucleotide Replacement 955 V. Comparison of Systems 956 VI. Concluding Remarks 957 VII. Acknowledgments 957 VIII. References 958

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A highly reproducible quantitative PCR (Q-PCR) assay was used to study the stability of human papillomavirus (HPV) in undifferentiated keratinocytes that maintain viral episomes. The term "stability" refers to the ability of episomes to persist with little copy number variation in cells. In investigating the mechanism of action of PA25, a previously published compound that destabilizes HPV episomes, aphidicolin was also found to markedly decrease episome levels, but via a different pathway from that of PA25. Since aphidicolin is known to activate DNA damage response (DDR) pathways, effects of inhibitors and small interfering RNAs (siRNAs) acting within DDR pathways were investigated. Inhibitors of Chk1 and siRNA directed against ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia Rad3-related (ATR) pathways significantly reduced viral episomes, suggesting that these pathways play a role in maintaining HPV episome stability. Inhibitors of Chk2 and DNA-PK had no effect on episome levels. Pharmacological inhibition of ATM proteins had no effect on episome levels, but ATM knockdown by siRNA significantly reduced episome levels, suggesting that ATM proteins are playing an important role in HPV episome stability that does not require kinase activity. These results outline two pathways that trigger episome loss from cells and suggest the existence of a littleunderstood mechanism that mediates viral DNA elimination. Together, our results also indicate that HPV episomes have a stability profile that is remarkably similar to that of fragile sites; these similarities are outlined and discussed. This close correspondence may influence the preference of HPV for integration into fragile sites.