Removal of cyclobutane pyrimidine dimers (CBPDs) in vivo from the DNA of UV-irradiated eight-leaf seedlings of Arabidopsis thaliana was rapid in the presence of visible light (half-life about 1 hour); removal of CBPDs in the dark, presumably via excision repair, was an order of magnitude slower. Extracts of plants contained significant photolyase in vitro, as assayed by restoration of transforming activity to UV-irradiated Escherichia coii plasmids; activity was maximal from four-leaf to 12-leaf stages. UV-B treatment of seedlings for 6 hours increased photolyase specific activity in extracts twofold. Arabidopsis photolyase was markedly temperature-sensitive, both in vitro (half-life at 300C about 12 minutes) and in vivo (half-life at 300C, 30 to 45 minutes).The wavelength dependency of the photoreactivation cross-section showed a broad peak at 375 to 400 nm, and is thus similar to that for maize pollen; it overlaps bacterial and yeast photolyase action spectra.Stratospheric ozone depletion projected for the early 21st century is expected to increase the amount of DNA-damaging UV-B (290-320 nm) radiation at the earth's surface by as much as 20 to 50% (2, 10, 33). This has renewed interest in the UV-resistance mechanisms of plants, which will be exposed continually to increased UV-B fluxes.Repair of ultraviolet-light-induced DNA damage is a feature of life at all levels of complexity. Studies of bacteria (particularly Escherichia coli), yeast, and mammalian cells have elucidated four distinct mechanisms by which cells cope with UV-damaged DNA (for reviews see refs. 9, 29). In photoreactivation, a single enzyme, photolyase, uses light energy in the 300 to 500 nm range to reverse a specific class of UV photoproducts-cis, syn CBPDs.2 In excision repair, an ensemble of proteins act together to remove a wide variety of helix-distorting lesions, including at least two UV photoproducts-CBPDs and pyrimidine-(6-4)-pyrimidone photoproducts (6-4 photoproducts). Photoproducts that block DNA replication are tolerated, rather than repaired,

The discovery that mutations in DNA mismatch repair genes can cause hereditary nonpolyposis colorectal cancer has stimulated interest in understanding the mechanism of DNA mismatch repair in eukaryotes. In the yeast Saccharomyces cerevisiae, DNA mismatch repair requires the MSH2, MLH1, and PMS1 proteins. Experiments revealed that the yeast MLH1 and PMS1 proteins physically associate, possibly forming a heterodimer, and that MLH1 and PMS1 act in concert to bind a MSH2-heteroduplex complex containing a G-T mismatch. Thus, MSH2, MLH1, and PMS1 are likely to form a ternary complex during the initiation of eukaryotic DNA mismatch repair.

The molecular pathogenesis of the myeloid leukemias that frequently occur in patients with Fanconi anemia (FA) is not well defined. Hematopoietic stem cells bearing inactivating mutations of FA complementation group C (FANCC) are genetically unstable and hypersensitive to apoptotic cytokine cues including IFN-gamma and TNF-alpha, but neoplastic stem cell clones that arise frequently in vivo are resistant to these cytokines. Reasoning that the combination of genetic instability and cytokine hypersensitivity might create an environment supporting the emergence of leukemic stem cells, we tested the leukemia-promoting effects of TNF-alpha in murine stem cells. TNF-alpha exposure initially profoundly inhibited the growth of Fancc-/- stem cells. However, longer-term exposure of these cells promoted the outgrowth of cytogenetically abnormal clones that, upon transplantation into congenic WT mice, led to acute myelogenous leukemia. TNF-alpha induced ROS-dependent genetic instability in Fancc-/- but not in WT cells. The leukemic clones were TNF-alpha resistant but retained their characteristic hypersensitivity to mitomycin C and exhibited high levels of chromosomal instability. Expression of FANCC cDNA in Fancc-/- stem cells protected them from TNF-alpha-induced clonal evolution. We conclude that TNF-alpha exposure creates an environment in which somatically mutated preleukemic stem cell clones are selected and from which unaltered TNF-alpha-hypersensitive Fancc-/- stem cells are purged.

The MutL protein is an essential component of the Escherichia coli methyl-directed mismatch repair system but has no known enzymatic function. In the yeast Saccharomyces cerevisiae, the MutL equivalent, an Mlh1p and Pms1p heterodimer, interacts with Msh2p bound to mismatch-containing DNA. Little is known of the functional domains of Mlh1p and Pms1p. In this report, we define the Mlh1p and Pms1p domains required for Mlh1p-Pms1p interaction. The Mlh1p-interactive domain of Pms1p is comprised of 260 amino acids near the carboxyl terminus while the Pms1p-interactive domain of Mlh1p resides in the final 212 residues. The two domains are sufficient for Mlh1p-Pms1p interaction, as determined by the two-hybrid assay and by in vitro protein affinity chromatography. Deletions within the domains completely eliminated Mlh1p-Pms1p interaction. Using site-directed mutagenesis, we altered a number of highly conserved residues in the Mlh1p and Pms1p proteins, including some alterations that mimic germline mutations observed for human hereditary nonpolyposis colorectal cancer. Alterations either in the consensus MutL box located in the amino-terminal portion of each protein or in the carboxyl-terminal homology motif of Mlh1p eliminated DNA mismatch repair function but had no effect on Mlh1p-Pms1p interaction. In addition, certain MLH1 and PMS1 mutant alleles caused a dominant negative mutator effect when overexpressed. We discuss the implications of these findings for the structural organization of the Mlh1p and Pms1p proteins and the importance of Mlh1p-Pms1p interaction.