A self-splicing group I intron has been found in the gene for a leucine transfer RNA in two species of Anabaena, a filamentous nitrogen-fixing cyanobacterium. The intron is similar to one that is found at the identical position in the same transfer RNA gene of chloroplasts of land plants. Because cyanobacteria were the progenitors of chloroplasts, it is likely that group I introns predated the endosymbiotic association of these eubacteria with eukaryotic cells.

DNA glycosylases are enzymes that perform the initial steps of base excision repair, the principal repair mechanism that identifies and removes endogenous damages that occur in an organism's DNA. We characterized the motion of single molecules of three bacterial glycosylases that recognize oxidized bases, Fpg, Nei, and Nth, as they scan for damages on tightropes of λ DNA. We find that all three enzymes use a key "wedge residue" to scan for damage because mutation of this residue to an alanine results in faster diffusion. Moreover, all three enzymes bind longer and diffuse more slowly on DNA that contains the damages they recognize and remove. Using a sliding window approach to measure diffusion constants and a simple chemomechanical simulation, we demonstrate that these enzymes diffuse along DNA, pausing momentarily to interrogate random bases, and when a damaged base is recognized, they stop to evert and excise it.DNA repair | search mechanisms O xidative DNA damage is produced endogenously during normal cellular metabolism or exogenously by chemical agents and ionizing radiation (1, 2). Oxidatively induced DNA damage resulting from attack by reactive oxygen species accounts for approximately one-half of all DNA base damages (3). Some oxidative base damages, such as thymine glycol, are blocks to DNA polymerases and thus potentially lethal; however, the majority of oxidative base lesions mispair with noncognate bases and are potentially mutagenic (4). Therefore, damaged bases must be repaired to maintain the cell's genomic integrity. With substantial in vivo steady-state levels of oxidative damages, alkylation damages, and apurinic/apyrimidinic (AP) sites among the nearly six billion normal bases, how DNA repair enzymes locate these damages in the sea of undamaged bases has been the subject of much speculation.The DNA repair mechanism responsible for the removal of the majority of endogenous DNA damages is the base excision repair (BER) pathway (4-6). The critical first step in BER is carried out by a DNA glycosylase that, fueled only by thermal energy, locates a damaged base and cleaves the N-glycosyl bond, thus removing the base lesion from the sugar phosphate backbone. Glycosylases are small monomeric proteins that are found in all living organisms and can be separated into different families based on substrate specificity and structural motifs. In Escherichia coli, there are three glycosylases, Nth, Fpg, and Nei, that directly remove oxidized DNA bases, and all three have an associated lyase activity that cleaves the DNA backbone. These glycosylases are members of two structural families, the helixhairpin-helix (HhH) or Nth superfamily and the helix-two turnshelix (H2TH) or Fpg/Nei family (7-9) (Fig. 1A). Interestingly, the HhH superfamily member, endonuclease III (Nth), and Fpg/ Nei family member, endonuclease VIII (Nei), primarily catalyze the removal of oxidized pyrimidines, such as 5,6-dihydroxy-5, 6-dihydrothymine (Tg), whereas formamidopyrimidine DNA glycosylase (Fpg) primarily removes oxidized purines...

Transcription and repair of many DNA helix-distorting lesions such as cyclobutane pyrimidine dimers have been shown to be coupled in cells across phyla from bacteria to humans. The signal for transcription-coupled repair appears to be a stalled transcription complex at the lesion site. To determine whether oxidative DNA lesions can block correctly initiated human RNA polymerase II, we examined the effect of site-specifically introduced oxidative damages on transcription in HeLa cell nuclear extracts. We found that transcription was blocked by single-stranded breaks, common oxidative DNA lesions, when present in the transcribed strand of the transcription template. Cyclobutane pyrimidine dimers, which have been previously shown to block transcription both in vitro and in vivo, also blocked transcription in the HeLa cell nuclear transcription assay. In contrast, the oxidative DNA base lesions, 8-oxoguanine, 5-hydroxycytosine, and thymine glycol did not inhibit transcription, although pausing was observed with the thymine glycol lesion. Thus, DNA strand breaks but not oxidative DNA base damages blocked transcription by RNA polymerase II.Transcription-coupled repair (TCR) 1 is a specialized form of DNA repair where damages are repaired preferentially in the transcribed strand of actively transcribed genes (for reviews, see Refs. 1 and 2). TCR was originally believed to be a subpathway of nucleotide excision repair; however, ionizing radiation damage (3), 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol; Tg) (4, 5), and 7,8-dihydro-8-oxoguanine (8-oxoG) (5) are removed in a TCR-dependent manner from human cells that lack nucleotide excision repair. Since Tg and 8-oxoG are small nonbulky lesions that are repaired primarily by the base excision repair (BER) pathway (for reviews, see Refs. 6 -8), TCR of Tg and 8-oxoG in cells that lack nucleotide excision repair (5) links TCR to BER. TCR of 8-oxoG has also been shown to occur in nonreplicating Escherichia coli cells (9). TCR of oxidative damage does not appear to be universal, since in Chinese hamster ovary cells, TCR of oxidative damage produced by photosensitization and oxidizing agents is not observed in the Dhfr and cFos genes (10 -12). Furthermore, DNA strand breaks, oxidative lesions also repaired by BER, do not appear to be repaired by TCR in the Dhfr gene from Chinese hamster ovary cells (13) or human colon cancer cells (14). Interestingly, recent measurements of TCR of cyclobutane pyrimidine dimers in the Hprt gene, integrated at different sites in Chinese hamster ovary cell chromosomes, have suggested that preferential repair of actively transcribed genes, as well as preferential repair of damages in the transcribed strand, is significantly affected by genomic context (15).The proposed signal for TCR is an RNA polymerase transcription complex stalled at a lesion, which recruits the repair proteins to the damage site (16 -18); the ability of a lesion on the transcribed strand to block the RNA polymerase transcription complex has been assumed to be crucial for T...

Many tRNA UAALeu genes from plastids contain a group I intron. An intron is also inserted in the same gene at the same position in cyanobacteria, the bacterial progenitors of plastids, suggesting an ancient bacterial origin for this intron. A group I intron has also been found in the tRNA fMet gene of some cyanobacteria but not in plastids, suggesting a more recent origin for this intron. In this study, we investigate the phylogenetic distributions of the two introns among cyanobacteria, from the earliest branching to the more derived species. The phylogenetic distribution of the tRNA UAA Leu intron follows the clustering of rRNA sequences, being either absent or present in clades of closely related species, with only one exception in the Pseudanabaena group. Our data support the notion that the tRNA UAA Leu intron was inherited by cyanobacteria and plastids through a common ancestor. Conversely, the tRNA fMet intron has a sporadic distribution, implying that many gains and losses occurred during cyanobacterial evolution. Interestingly, a phylogenetic tree inferred from intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history.Ever since their discovery, the origin of introns has been a subject of controversy. One view, the introns-late hypothesis, proposes that introns are recent invaders and that split genes arose by late insertion of introns into originally uninterrupted genes (28). In that scenario, horizontal transfer and transposition of introns are frequent events, accounting for the scattered phylogenetic distribution of introns. Although the debate has focused on spliceosomal introns, such a scenario could apply as well to other types of introns, some of which are known to be mobile (22). In contrast, the introns-early view implies that introns are very ancient, being present in the progenote (universal ancestor) (7). The demonstration that some members of group I and group II introns are capable of in vitro autocatalytic activity (19,29,39) lends further support to the presence of these introns at an early stage of evolution, maybe as early as the putative precellular RNA world (13). In such a scenario, the observed phylogenetic distribution of introns could be explained by multiple losses in different lineages during evolution (7) and by their mobility, which is assumed to be a derived feature (2). A major obstacle for the introns-early hypothesis was the apparent absence of introns in eubacteria, although this was tentatively rationalized by pressure to streamline the genome in rapidly dividing bacteria (7). Discovery of group I introns in bacteriophages of gram-positive and gram-negative bacteria did not help to resolve the issue, due to uncertainties concerning the origin of the bacteriophages themselves (see discussion in reference 35). The recent discovery of both group I and group II introns in divergent eubacteria (4,11,12,20,31,44) was acclaimed as a breakthrough by introns-early proponents. In most cases, however, the relatio...

Purpose: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cellfree in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data. Methods: Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories. Results: Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible. Conclusion: The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants.