Heterozygous bacteriophage A DNA molecules, whose replication requires mismatch correction of a mutant nucleotide in the transcribed strand, provide an assay for localized mismatch repair in Escherichia coli. We describe two systems: one removes the A in C-A or G-A mismatches and the other removes one or the other C in a C C mismatch. Mutations disabling the first system result in a mutator phenotype that may be identical to mutY. Investigation of the products present in infective centers of bacteria transfected with artificially constructed heteroduplex DNA molecules of bacteriophage A has provided evidence for the mode of action of two mismatch repair systems in E. coli (5, 6). One of these, the adenine methylationdirected mismatch repair system, has been shown to play an important role in replication fidelity by repairing errors in a newly replicated DNA strand, distinguishing that strand by the absence of methylation at GATC sequences. This repair process directs the replacement of long tracts of the undermethylated strand (several kilobases) when a base pair mismatch is present (7). Some of the functions repaired for this mismatch repair system are encoded by mutL, mutS, mutH, and mutU (uvrD) (8). A localized repair activity became evident when the methyl-directed mismatch repair system was disabled as a result of mutations in mutH or mutU (5). This localized repair results in separation of very closely linked markers, requires the functions mutL and mutS, is independent of adenine methylation, efficiently corrects the T of G-T mismatches resulting from a C to T transition mutation in the inner C in CC(A/T)G sites (6), and is similar to the very short patch repair described by Lieb (9, 10).Serendipitous evidence of additional localized mismatch repair (11) suggested a strategy that allows the investigation of repair capacities, one mismatch at a time. DNA is isolated from phage harboring conditional (amber) mutations in functions essential for DNA replication, the strands are separated, and complementary strands from each of two different mutants are hybridized. The heteroduplex molecules are packaged in vitro and the assembled phage can be plated under permissive conditions, to determine the phage yield, or on hosts that do not suppress the amber mutations. In the latter case, plaque formation requires repair of the mutant nucleotide on the transcribed strand, without co-correction of the neighboring wild-type nucleotide that is, in turn, mismatched with a mutant nucleotide on the untranscribed strand. Thus, plaque formation reflects repair of a particular mismatch. This strategy has revealed the presence of two mismatch repair capabilities present in E. coli. One of these functions removes the A of C-A and G-A mismatches and the other corrects a C in a C C mismatch. When the A removal function is disabled, the bacteria display a mutator phenotype.

SummaryIn C. elegans, the skn‐1 gene encodes a transcription factor that resembles mammalian Nrf2 and activates a detoxification response. skn‐1 promotes resistance to oxidative stress (Oxr) and also increases lifespan, and it has been suggested that the former causes the latter, consistent with the theory that oxidative damage causes aging. Here, we report that effects of SKN‐1 on Oxr and longevity can be dissociated. We also establish that skn‐1 expression can be activated by the DAF‐16/FoxO transcription factor, another central regulator of growth, metabolism, and aging. Notably, skn‐1 is required for Oxr but not increased lifespan resulting from over‐expression of DAF‐16; concomitantly, DAF‐16 over‐expression rescues the short lifespan of skn‐1 mutants but not their hypersensitivity to oxidative stress. These results suggest that SKN‐1 promotes longevity by a mechanism other than protection against oxidative damage.

Exposure of eukaryotic cells to UV light induces a checkpoint response that delays cell-cycle progression after cells enter S phase. It has been hypothesized that this checkpoint response provides time for repair by signaling the presence of structures generated when the replication fork encounters UV-induced DNA damage. To gain insight into the nature of the signaling structures, we used time-lapse microscopy to determine the effects of deficiencies in translesion DNA polymerases on the checkpoint response of the fission yeast Schizosaccharomyces pombe. We found that disruption of the genes encoding translesion DNA polymerases Polκ and Polη significantly prolonged the checkpoint response, indicating that the substrates of these enzymes are signals for checkpoint activation. Surprisingly, we found no evidence that the translesion polymerases Rev1 and Polζ repair structures that are recognized by the checkpoint despite their role in maintaining viability after UV irradiation. Quantitative flow cytometry revealed that cells lacking translesion polymerases replicate UV-damaged DNA at the same rate at WT cells, indicating that the enhanced checkpoint response of cells lacking Polκ and Polη is not the result of stalled replication forks. These observations support a model in which postreplication DNA gaps with unrepaired UV lesions in the template strand act both as substrates for translesion polymerases and as signals for checkpoint activation.NA damage produced by the UV component of sunlight presents a constant challenge for the survival of organisms on the earth's surface. In response to this challenge, eukaryotic cells have evolved excision repair processes that remove the damage, postreplication repair processes that facilitate the replication of damaged DNA, and checkpoint mechanisms that delay cell-cycle progression to make time for repair (1-3). Because the genes that mediate these processes are conserved in eukaryotes, model systems such as yeast have provided valuable insights that are applicable to the DNA damage responses of higher organisms (4). In previous studies, we used time-lapse microscopy to measure the cell-cycle dynamics of UV-irradiated fission yeast cells and observed two distinct DNA damage checkpoint responses: the previously characterized G2/M checkpoint response that delays cell division when cells are irradiated in G2 phase and a postreplication checkpoint response that delays division when cells irradiated in any stage of the cell cycle carry lesions into S phase (5). Only the latter response occurs after moderate UV doses comparable to sunlight exposure, so it is likely to be particularly important in nature (3). The postreplication checkpoint response is activated following the encounter of replication forks with UV-induced DNA damage and requires the activity of checkpoint proteins that recognize structures containing transitions between double-stranded and single-stranded DNA (3, 6-11). The nature and origin of the signaling structures that determine the Schizosaccharomyces pombe po...

The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, β and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK β subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved.