Mitochondrial DNA damage and animal longevity: insights from comparative studies

Chemical reactions in living cells are under strict enzyme control and conform to a tightly regulated metabolic program. However, uncontrolled and potentially deleterious endogenous reactions occur, even under physiological conditions. Aging, in this chemical context, could be viewed as an entropic process, the result of chemical side reactions that chronically and cumulatively degrade the function of biological systems. Mitochondria are a main source of reactive oxygen species (ROS) and chemical sidereactions in healthy aerobic tissues and are the only known extranuclear cellular organelles in animal cells that contain their own DNA (mtDNA). ROS can modify mtDNA directly at the sugar-phosphate backbone or at the bases, producing many different oxidatively modified purines and pyrimidines, as well as single and double strand breaks and DNA mutations. In this scenario, natural selection tends to decrease the mitochondrial ROS generation, the oxidative damage to mtDNA, and the mitochondrial mutation rate in long-lived species, in agreement with the mitochondrial oxidative stress theory of aging.

Sequencing human-gibbon breakpoints of synteny reveals mosaic new insertions at rearrangement sites

The gibbon genome exhibits extensive karyotypic diversity with an increased rate of chromosomal rearrangements during evolution. In an effort to understand the mechanistic origin and implications of these rearrangement events, we sequenced 24 synteny breakpoint regions in the white-cheeked gibbon (Nomascus leucogenys, NLE) in the form of high-quality BAC insert sequences (4.2 Mbp). While there is a significant deficit of breakpoints in genes, we identified seven human gene structures involved in signaling pathways (DEPDC4, GNG10), phospholipid metabolism (ENPP5, PLSCR2), beta-oxidation (ECH1), cellular structure and transport (HEATR4), and transcription (ZNF461), that have been disrupted in the NLE gibbon lineage. Notably, only three of these genes show the expected evolutionary signatures of pseudogenization. Sequence analysis of the breakpoints suggested both nonclassical nonhomologous end-joining (NHEJ) and replication-based mechanisms of rearrangement. A substantial number (11/24) of human-NLE gibbon breakpoints showed new insertions of gibbon-specific repeats and mosaic structures formed from disparate sequences including segmental duplications, LINE, SINE, and LTR elements. Analysis of these sites provides a model for a replication-dependent repair mechanism for double-strand breaks (DSBs) at rearrangement sites and insights into the structure and formation of primate segmental duplications at sites of genomic rearrangements during evolution.

Assessment of microbial community structure changes by amplified ribosomal DNA restriction analysis (ARDRA)

Amplified ribosomal DNA restriction analysis (ARDRA) is a simple method based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In this study we have evaluated the suitability of this method to detect differences in activated sludge bacterial communities fed on domestic or industrial wastewater, and subject to different operational conditions. The ability of ARDRA to detect these differences has been tested in modified Ludzack-Ettinger (MLE) configurations. Samples from three activated sludge wastewater treatment plants (WWTPs) with the MLE configuration were collected for both oxic and anoxic reactors, and ARDRA patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice similarity coefficients was calculated and used to compare these restriction patterns. Differences in the community structure due to influent characteristics and temperature could be observed, but not between the oxic and anoxic reactors of each of the three MLE configurations. Other possible applications of ARDRA for detecting and monitoring changes in activated sludge systems are also discussed

Counter-ion effect on surfactant-DNA gel particles as controlled DNA delivery systems

The ability to entrap drugs within vehicles and subsequently release them has led to new treatments for a number of diseases. Based on an associative phase separation and interfacial diffusion approach, we developed a way to prepare DNA gel particles without adding any kind of cross-linker or organic solvent. Among the various agents studied, cationic surfactants offered particularly efficient control for encapsulation and DNA release from these DNA gel particles. The driving force for this strong association is the electrostatic interaction between the two components, as induced by the entropic increase due to the release of the respective counter-ions. However, little is known about the influence of the respective counter-ions on this surfactant-DNA interaction. Here we examined the effect of different counter-ions on the formation and properties of the DNA gel particles by mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with the single chain surfactant dodecyltrimethylammonium (DTA). In particular, we used as counter-ions of this surfactant the hydrogen sulfate and trifluoromethane sulfonate anions and the two halides, chloride and bromide. Effects on the morphology of the particles obtained, the encapsulation of DNA and its release, as well as the haemocompatibility of these particles, are presented, using the counter-ion structure and the DNA conformation as controlling parameters. Analysis of the data indicates that the degree of counter-ion dissociation from the surfactant micelles and the polar/hydrophobic character of the counter-ion are important parameters in the final properties of the particles. The stronger interaction with amphiphiles for ssDNA than for dsDNA suggests the important role of hydrophobic interactions in DNA.

Current directions in DNA gel particles

A general understanding of interactions between DNA andoppositely charged compounds forms the basis for developing novelDNA-based materials, including gel particles. The association strength,which is altered by varying the chemical structure of the cationiccosolute, determines the spatial homogeneity of the gelation process,creating DNA reservoir devices and DNA matrix devices that can bedesigned to release either single- (ssDNA) or double-stranded(dsDNA) DNA. This paper reviews the preparation of DNA gelparticles using surfactants, proteins and polysaccharides. Particlemorphology, swelling/dissolution behaviour, degree of DNAentrapment and DNA release responses as a function of the nature ofthe cationic agent used are discussed. Current directions in thehaemocompatible and cytotoxic characterization of these DNA gelparticles have been also included.

Substrate-selective repair and restart of replication forks by DNA translocases

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.