Prebiotic Phosphorylation and Concomitant Oligomerization of Deoxynucleosides to form DNA.

Recent demonstrations of RNA-DNA chimeras (RDNA) enabling RNA and DNA replication, coupled with prebiotic co-synthesis of deoxyribo- and ribo-nucleotides, have resurrected the hypothesis of co-emergence of RNA and DNA. As further support, we show that diamidophosphate (DAP) with 2-aminoimidazole (amido)phosphorylates and oligomerizes deoxynucleosides to form DNA-under conditions similar to those of ribonucleosides. The pyrimidine deoxynucleoside 5'-O-amidophosphates are formed in good (≈60 %) yields. Intriguingly, the presence of pyrimidine deoxynucleos(t)ides increased the yields of purine deoxynucleotides (≈20 %). Concomitantly, oligomerization (≈18-31 %) is observed with predominantly 3',5'-phosphodiester DNA linkages, and some (<5 %) pyrophosphates. Combined with previous observations of DAP-mediated chemistries and the constructive role of RDNA chimeras, the results reported here help set the stage for systematic investigation of a systems chemistry approach of RNA-DNA coevolution.

Highly stable single-strand-specific 3'-nuclease/nucleotidase from Legionella pneumophila.

The Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1-P1 type nuclease. Bacterial zinc-dependent 3'-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1 (Lpn1), in contrast to many eukaryotic representatives with in-depth studies available. To help explain the principle properties and role of these enzymes in intracellular prokaryotic pathogens we have designed and optimized a heterologous expression protocol utilizing E. coli together with an efficient purification procedure, and performed detailed characterization of the enzyme. Replacement of Ni2+ ions by Zn2+ ions in affinity purification proved to be a crucial step in the production of pure and stable protein. The production protocol provides protein with high yield, purity, stability, and solubility for structure-function studies. We show that highly thermostable Lpn1 is active mainly towards RNA and ssDNA, with pH optima 7.0 and 6.0, respectively, with low activity towards dsDNA; the enzyme features pronounced substrate inhibition. Bioinformatic and experimental analysis, together with computer modeling and electrostatics calculations point to an unusually high positive charge on the enzyme surface under optimal conditions for catalysis. The results help explain the catalytic properties of Lpn1 and its substrate inhibition.