A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis

During a systematic analysis of conserved gene context in prokaryotic genomes, a previously undetected, complex, partially conserved neighborhood consisting of more than 20 genes was discovered in most Archaea (with the exception of Thermoplasma acidophilum and Halobacterium NRC-1) and some bacteria, including the hyperthermophiles Thermotoga maritima and Aquifex aeolicus. The gene composition and gene order in this neighborhood vary greatly between species, but all versions have a stable, conserved core that consists of five genes. One of the core genes encodes a predicted DNA helicase, often fused to a predicted HD-superfamily hydrolase, and another encodes a RecB family exonuclease; three core genes remain uncharacterized, but one of these might encode a nuclease of a new family. Two more genes that belong to this neighborhood and are present in most of the genomes in which the neighborhood was detected encode, respectively, a predicted HD-superfamily hydrolase (possibly a nuclease) of a distinct family and a predicted, novel DNA polymerase. Another characteristic feature of this neighborhood is the expansion of a superfamily of paralogous, uncharacterized proteins, which are encoded by at least 20–30% of the genes in the neighborhood. The functional features of the proteins encoded in this neighborhood suggest that they comprise a previously undetected DNA repair system, which, to our knowledge, is the first repair system largely specific for thermophiles to be identified. This hypothetical repair system might be functionally analogous to the bacterial–eukaryotic system of translesion, mutagenic repair whose central components are DNA polymerases of the UmuC-DinB-Rad30-Rev1 superfamily, which typically are missing in thermophiles.     

The gene encoding translation initiation factor 3 is highly conserved in gram-negative bacteria

A 1.1-kb Hp alpha I fragment of the Escherichia coli chromosome containing the gene for translation initiation factor 3 was employed as a probe in heterologous hybridization to chromosomal DNA from a variety of other procaryotes. Positive hybridization was observed to DNA derived from all gram-negative bacteria tested. In contrast, no hybridization to DNA from gram-positive bacteria was detected. In addition, homologous sequences were found in Euglena gracilis chloroplast DNA, while this was not the case with Saccharomyces cerevisiae mitochondrial DNA. These results are discussed in light of existing data on the components and mechanism of translation initiation in the various organisms and organelles employed in this study.     

Biotin sensing in Saccharomyces cerevisiae is mediated by a conserved DNA element and requires the activity of biotin-protein ligase

Biotin is a water-soluble vitamin that functions as a prosthetic group in carboxylation reactions. In addition to its role as a cofactor, biotin has multiple roles in gene regulation. We analyzed biotin effects on gene expression in the yeast Saccharomyces cerevisiae and demonstrated by microarray, Northern, and Western analyses that all yeast genes encoding proteins involved in biotin metabolism are up-regulated following biotin depletion. Many of these genes contain a palindromic promoter element that is necessary and sufficient for mediating the biotin response and functions as an upstream-activating sequence. Mutants lacking the plasma membrane biotin transporter Vht1p display constitutively high expression levels of biotin-responsive genes. However, they react normally to biotin precursors that do not require Vht1p for uptake. The biotin-like effect of precursors with regard to gene expression requires their intracellular conversion to biotin. This demonstrates that Vht1p does not act as a sensor for biotin and that intracellular biotin is crucial for gene expression. Mutants with defects in biotin-protein ligase, similar to vht1delta mutants, also display aberrantly high expression of biotin-responsive genes. Like vht1delta cells, they have reduced levels of protein biotinylation, but unlike vht1delta mutants, they possess normal levels of free intracellular biotin. This indicates that free intracellular biotin is irrelevant for gene regulation and identifies biotin-protein ligase as an important element of the biotin-sensing pathway in yeast.     

Oxidation by DNA charge transport damages conserved sequence block II, a regulatory element in mitochondrial DNA

Sites of oxidative damage in mitochondrial DNA have been identified on the basis of DNA-mediated charge transport. Our goal is to understand which sites in mitochondrial DNA are prone to oxidation at long range and whether such oxidative damage correlates with cancerous transformation. Here we show that a primer extension reaction can be used to monitor directly oxidative damage to authentic mitochondrial DNA through photoreactions with a rhodium intercalator. The complex [Rh(phi)2bpy]Cl3 (phi = 9,10-phenanthrenequinone diimine) binds to DNA without sequence specificity and, upon photoactivation, either promotes strand breaks directly at the binding site or promotes one-electron oxidative damage; comparing the sites of base oxidation to direct strand breaks reveals the oxidative damage that arises from a distance through DNA-mediated charge transport. Significantly, base oxidation by charge transport overlaps with known mutational hot spots associated with cancers at nucleotides surrounding positions 263 and 303; the latter is known as conserved sequence block II and is vital to DNA replication. Since DNA base oxidation at conserved sequence block II should weaken the ability of damaged mitochondrial genomes to be replicated, DNA-mediated charge transport may provide a protection mechanism for excluding damaged DNA.