Introns are common in the rRNA gene loci of fungal genomes, but biochemical studies to investigate splicing are rare. Here, self-splicing of a very small (67 nucleotide) group I intron is demonstrated. The PaSSU intron (located within the rRNA small subunit gene of Phialophora americana) splices in vitro under group I intron conditions. Most group I ribozymes contain pairing regions P1-P10, with a conserved G.U pair at the 5' splice site, and a G at the 3' intron border. The PaSSU intron contains only P1, P7, and P10. While it contains the G.U pair at the 5' splice, a U is found at the 3' end of the intron instead of a G. Phylogenetic analysis places it within subgroup IC1, whose members are found in the nuclear rRNA genes of fungi. The structural elements are similar to those in the centermost regions of other group I introns. Its size can be explained by a single large deletion that removed P2 through much of P9. Part of the original P9 region has assumed the function of P7. Its small size and genealogy makes it an excellent model to study RNA catalysis and evolution.

BackgroundGenome instability is associated with human cancers and chromosome breakage syndromes, including Bloom's syndrome, caused by inactivation of BLM helicase. Numerous mutations that lead to genome instability are known, yet how they interact genetically is poorly understood.ResultsWe show that spontaneous translocations that arise by nonallelic homologous recombination in DNA-damage-checkpoint-defective yeast lacking the BLM-related Sgs1 helicase (sgs1Δ mec3Δ) are inhibited if cells lack Mec1/ATR kinase. Tel1/ATM, in contrast, acts as a suppressor independently of Mec3 and Sgs1. Translocations are also inhibited in cells lacking Dun1 kinase, but not in cells defective in a parallel checkpoint branch defined by Chk1 kinase. While we had previously shown that RAD51 deletion did not inhibit translocation formation, RAD59 deletion led to inhibition comparable to the rad52Δ mutation. A candidate screen of other DNA metabolic factors identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1Δ mutant, becoming even more important for chromosomal stability upon MEC3 deletion. We determined that the C-terminal third of Exo1, harboring mismatch repair protein binding sites and phosphorylation sites, is dispensable for Exo1's roles in chromosomal rearrangement suppression, mutation avoidance and resistance to DNA-damaging agents.ConclusionsOur findings suggest that translocations between related genes can form by Rad59-dependent, Rad51-independent homologous recombination, which is independently suppressed by Sgs1, Tel1, Mec3 and Exo1 but promoted by Dun1 and the telomerase-inhibitor Mec1. We propose a model for the functional interaction between mitotic recombination and the DNA-damage checkpoint in the suppression of chromosomal rearrangements in sgs1Δ cells.

Rev3 polymerase and Mph1 DNA helicase participate in error-prone and error-free pathways, respectively, for the bypassing of template lesions during DNA replication. Here we have investigated the role of these pathways and their genetic interaction with recombination factors, other nonreplicative DNA helicases, and DNA damage checkpoint components in the maintenance of genome stability, viability, and sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). We find that cells lacking Rev3 and Mph1 exhibit a synergistic, Srs2-dependent increase in the rate of accumulating spontaneous, gross chromosomal rearrangements, suggesting that the suppression of point mutations by deletion of REV3 may lead to chromosomal rearrangements. While mph1⌬ is epistatic to homologous recombination (HR) genes, both Rad51 and Rad52, but not Rad59, are required for normal growth of the rev3⌬ mutant and are essential for survival of rev3⌬ cells during exposure to MMS, indicating that Mph1 acts in a Rad51-dependent, Rad59-independent subpathway of HR-mediated lesion bypass. Deletion of MPH1 helicase leads to synergistic DNA damage sensitivity increases in cells with chl1⌬ or rrm3⌬ helicase mutations, whereas mph1⌬ is hypostatic to sgs1⌬. Previously reported slow growth of mph1⌬ srs2⌬ cells is accompanied by G 2 /M arrest and fully suppressed by disruption of the Mec3-dependent DNA damage checkpoint. We propose a model for replication fork rescue mediated by translesion DNA synthesis and homologous recombination that integrates the role of Mph1 in unwinding D loops and its genetic interaction with Rev3 and Srs2-regulated pathways in the suppression of spontaneous genome rearrangements and in mutation avoidance.Nonreplicative DNA helicases play an important role in the maintenance of genome stability from bacteria to humans, most likely by affecting the formation and/or resolution of recombination intermediates and by facilitating replication fork progression through chromosomal regions with a propensity to adopt unusual DNA structures or those bound by proteins. In Saccharomyces cerevisiae, this group of DNA helicases includes the 3Ј-to-5Ј helicases Sgs1 and Srs2 and the 5Ј-to-3Ј DNA helicase Rrm3. In the absence of any two of these three helicases, unresolved recombination intermediates accumulate and lead to extremely slow growth that is fully suppressed by deletion of genes encoding early homologous recombination (HR) factors (4,6,17,20,37,46). In the absence of Sgs1, cells exhibit increased rates of mitotic recombination, frequent chromosome missegregation, accumulation of extrachromosomal ribosomal DNA (rDNA) circles, and increased rates of gross chromosomal rearrangements (GCRs) involving nonhomologous chromosomes (5,24,25,38,40,43,49,50). Based on the increased crossover frequency during HO endonucleaseinduced double-strand breaks (DSBs) in cells lacking Sgs1, it has also been proposed that Sgs1 may function in decatenation of Holliday junctions (HJs) to yield noncrossovers (12,22). Like Sgs1, Srs2 acts to favor noncro...

BackgroundGroup I introns (specifically subgroup IC1) are common in the nuclear ribosomal RNA genes of fungi. While most range in length from more than 200 to nearly 1800 nucleotides (nt) in length, several small putative (or degenerate) group I introns have been described that are between 56 and 81 nt. Although small, previously we demonstrated that the PaSSU intron in the rRNA small subunit gene of Phialophora americana isolate Wang 1046 is capable of in vitro splicing using a standard group I intron pathway, thus qualifying it as a functional ribozyme.FindingsHere, we describe eight short putative group I introns, ranging in length from 63 to 75 nt, in the rRNA small subunit genes of Phialophora isolates, a fungal genus that ranges from saprobic to pathogenic on plants and animals. All contain putative pairing regions P1, P7, and P10, as well as a pairing region formed between the middle of the intron and part of the 3' exon. The other pairing regions common in the core of standard group I introns are absent. However, parts of the 3' exon may aid in the stabilization of these small introns. Although the eight putative group I introns were from at least three species of Phialophora, phylogenetic analysis indicated that the eight are monophyletic. They are also monophyletic with the small introns of two lichen-forming fungi, Porpidia crustulata and Arthonia lapidicola.ConclusionsThe small putative group I introns in Phialophora have common features that may represent group I introns at their minima. They appear to have a single origin as indicated by their monophyly in phylogenetic analyses.