Complex dynamics in a cross-catalytic self-replication mechanism.

The authors consider a minimal cross-catalytic self-replicating system of only two cross-catalytic templates that mimics the R3C ligase ribozyme system of Dong-Eu and Joyce [Chem. Biol. 11, 1505 (2004)]. This system displays considerably more complex dynamics than its self-replicating counterpart. In particular, the authors discuss the Poincare-Andronov-Hopf bifurcation, canard transitions, excitability, and hysteresis that yield birhythmicity between simple and complex oscillations.

Structure-function analysis of T4 RNA ligase 2.

Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a polynucleotide ligase family that includes the trypanosome RNA-editing ligases and putative RNA ligases encoded by eukaryotic viruses and archaea. Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining. We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of ligase adenylylation and phosphodiester bond formation. The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates. Our mutagenesis results underscore a shared active site architecture in Rnl2-like ligases, DNA ligases, and mRNA capping enzymes. They also highlight two essential signature residues, Glu(34) and Asn(40), that flank the active site lysine nucleophile (Lys(35)) and are unique to the Rnl2-like ligase family.

Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains.

RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA ligase and trypanosome RNA-editing ligases. Here, we identified another RNA ligase from the bacterial domain--a second RNA ligase (Rnl2) encoded by phage T4. Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through ligase-adenylate and RNA-adenylate intermediates. Mutational analysis identifies amino acids required for the ligase-adenylation or phosphodiester synthesis steps of the ligation reaction. The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA ligases and RNA capping enzymes. Rnl2 has scant amino acid similarity to T4 gp63. Rather, Rnl2 exemplifies a distinct ligase family, defined by variant motifs, that includes the trypanosome-editing ligases and a group of putative RNA ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea. These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.

UV-induced mutagenesis of phage S13 can occur in the absence of the RecA and UmuC proteins of Escherichia coli.

The UV-induced mutagenesis of phage S13 that accompanies Weigle repair is known to require the products of the recA and umuDC genes, as does the UV-induced mutagenesis of the Escherichia coli chromosome. I found that UV-induced mutagenesis of phage S13 occurred in the absence of both the RecA and UmuC functions when the irradiated phage was photoreactivated. Furthermore, UV-induced phage mutations were produced in a recA- umuC- cell even without photoreactivation and in the absence of any other known UV repair mechanism, at a frequency 29% of that found after photoreactivation and 7% of that found after Weigle repair, implying that DNA synthesis can proceed past a dimer at an unexpectedly high frequency even when unaided by the UmuC-RecA SOS repair functions. The unaided DNA synthesis appears capable of producing mutations in the vicinity of a pyrimidine dimer; by aiding synthesis past a dimer, a repair mechanism may disclose a mutation without having any active role in producing it.

The cdc9 ligase joins completed replicons in baker's yeast.

The drug hydroxyurea has been found to affect the conditional DNA ligase mutant cdc9 in the same way as a wild type. Specific concentrations inhibit the joining of completed replicons leading to a substantial accumulation of these molecules. Upon removal of hydroxyurea and further incubation of cdc9 cells at the permissive temperature the replicons joined together, while in sharp contrast at the restrictive temperature no such joining occurred. However, a revertant of cdc9 able to grow at the restrictive temperature was also able to join replicons under these conditions, so the cdc9 ligase must be responsible for the assembly of completed replicons.