A shared DNA-damage-response pathway for induction of stem-cell death by UVB and by gamma irradiation.

Both UVB radiation and DNA-breaking agents were previously reported to kill Arabidopsis stem cells. We demonstrate that death induced by UVB or by ionizing radiation (IR) requires Suppressor of Gamma Response 1 (SOG1), a transcription factor already found to govern many responses to these agents in Arabidopsis. DNA-damage responses (DDRs) triggered primarily by replication-blocking photoadducts or double-strand-breaks thus converge to a shared programmed-cell-death (PCD) pathway. Both UVB- and IR-induced PCD also require functional DDR protein kinases. Employment of atr atm mutants (uniquely available in Arabidopsis) shows that either ATR (which recognizes ssDNA) or ATM (which recognizes DSBs) suffices for PCD induction by either agent. Thus, DNA damage made by UVB or by IR engenders both ATM-activating and ATR-activating structures. The elevated PCD in UVB-irradiated atr and atm mutants suggests that in wt plants ATR and/or ATM may activate both pathways that avert PCD and those that elicit it. The similar PCD levels induced by roughly 30,000 unrepaired photoadducts vs. 20 IR-induced DSBs indicate that DDR damage-tolerance activities in this model stem-cell niche are remarkably efficient.

Simple and rapid preparation of gapped plasmid DNA for incorporation of oligomers containing specific DNA lesions.

The cytotoxicity, mutagenicity, and carcinogenicity of DNA base lesions are largely determined by the responses of cellular DNA repair proteins, DNA polymerases, and signaling pathways. Elucidation of these processes is thus of high biochemical interest. Such studies increasingly rely on DNA substrates containing specific lesions at defined locations. Although short synthetic DNA oligomers have frequently proved useful, circular plasmid substrates are preferable for much biochemical work, and essential for in vivo studies. However, the complexity of current approaches for preparing such substrates and limitations inherent in the procedures have posed problems. We present here a simple, highly versatile procedure for preparing gapped duplex plasmids, into which oligomers incorporating specific lesions can easily be inserted. Endonuclease N.BstNBI was used to nick twice the same strand of a pUC19-derived plasmid (pUC19HBDa), at two GAGTCNNNN sequences separated by 22 bases. Removal of the 22-nt oligomer and further purification produced a highly pure gapped plasmid. To illustrate application of this procedure, 22-nt oligonucleotides containing a single uracil residue were ligated into the gapped molecules. The pUC19HB(Da) plasmid can be modified to accept almost any DNA-lesion-containing oligomer. Using this new approach to incorporate specific DNA lesions into popular reporter genes will facilitate in vivo study of cellular responses to DNA damage.

Replication of chloroplast, mitochondrial and nuclear DNA during growth of unirradiated and UVB-irradiated Arabidopsis leaves.

Replication of Arabidopsis nuclear, mitochondrial and chloroplast DNA (ncDNA, mtDNA, cpDNA) was assayed by measuring respective changes in copies per leaf, employing quantitative PCR (QPCR) analysis with genome-specific primer pairs. All three genomes showed parallel increases during growth of cotyledons and 5th leaves in planta, maintaining approximately 13 mtDNA copies and 280 cpDNA copies per haploid nuclear genome. Detached 5th leaves, which showed good growth and DNA replication on agar plates, were irradiated at (DNA-effective) UV-B fluences of 1.3-5.0 kJ m-2 and incubated under blue (photorepair-active) plus gold light or gold light only. Under blue light, replication of all genomes after all UV fluences was approximately as efficient as replication in unirradiated leaves. UV-irradiated leaves showed little growth under gold light only; 5 kJ m-2 stopped replication of all three genomes, 2.5 kJ m-2 stopped only cpDNA replication, and 1.3 kJ m-2 only delayed cpDNA replication. Immunoassays showed that 5 kJ m-2 induced about 1.2 cyclobutane pyrimidine dimers and 0.1 [6-4]photoproducts per kbp of bulk DNA, and that both photoproducts were completely removed during 2-3 days under blue light, suggesting efficient photorepair of at least ncDNA and cpDNA. The evidence for efficient photorepair of organellar DNA contrasts with previous studies of irradiated 5-day-old seedlings, and with the apparent absence of Arabidopsis photolyases bearing transit peptides.

Preparation of DNA substrates for in vitro mismatch repair.

Analyses in vitro of correction of DNA mismatches have been pivotal in biochemical dissection of mismatch repair pathways. However, the complex procedures needed to prepare DNA substrates for mismatch repair have posed substantial barriers to investigators who wish to pursue such analyses. Here we describe a simple, efficient way to prepare a variety of mismatched DNA substrates. We use in our procedure high-copy-number pUC19-derived plasmids, and a newly commercially available endonuclease N.BstNBI that makes site-specific single-strand nicks. The ability to prepare large substrate quantities in a relatively short time and to construct wider ranges of different mismatches in various sequence contexts will facilitate future research.

Arabidopsis MutS homologs-AtMSH2, AtMSH3, AtMSH6, and a novel AtMSH7-form three distinct protein heterodimers with different specificities for mismatched DNA.

Arabidopsis mismatch repair genes predict MutS-like proteins remarkably similar to eukaryotic MutS homologs-MSH2, MSH3, and MSH6. A novel feature in Arabidopsis is the presence of two MSH6-like proteins, designated AtMSH6 and AtMSH7. Combinations of Arabidopsis AtMSH2 with AtMSH3, AtMSH6, or AtMSH7 proteins-products of in vitro transcription and translation-were analyzed for interactions by analytical gel filtration chromatography. The AtMSH2 protein formed heterodimers with AtMSH3, AtMSH6, and AtMSH7, but no single proteins formed homodimers. The abilities of the various heterodimers to bind to mismatched 51-mer duplexes were measured by electrophoretic mobility-shift assays. Similar to the behavior of the corresponding human proteins, AtMSH2*AtMSH3 heterodimers bound "insertion-deletion" DNA with three nucleotides (+AAG) or one nucleotide (+T) looped out much better than they bound DNA with a base/base mispair (T/G), whereas AtMSH2*AtMSH6 bound the (+T) substrate strongly, (T/G) well, and (+AAG) no better than it did a (T/A) homoduplex. However, AtMSH2*AtMSH7 showed a different specificity: moderate affinity for a (T/G) substrate and weak binding of (+T). Thus, AtMSH2*AtMSH7 may be specialized for lesions/base mispairs not tested or for (T/G) mispairs in special contexts.

Antagonism of ultraviolet-light mutagenesis by the methyl-directed mismatch-repair system of Escherichia coli.

Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2.MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to "matched" photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC --> CTC and CTT --> CTC transitions. F' lacZ targets were mated from mut(+) donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu(+) mut(+) recipients, a range of UV fluences induced lac(+) revertant frequencies of 4-25 x 10(-8); these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd(-) defect, it appears not to involve transcription-coupled excision repair. In mut(+) umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m(2)) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5-10 x 10(-8). Thus, at UV doses too low to induce SOS functions, such as Umu(2)'D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial.

Evolutionary origin, diversification and specialization of eukaryotic MutS homolog mismatch repair proteins.

Most eubacteria, and all eukaryotes examined thus far, encode homologs of the DNA mismatch repair protein MutS. Although eubacteria encode only one or two MutS-like proteins, eukaryotes encode at least six distinct MutS homolog (MSH) proteins, corresponding to conserved (orthologous) gene families. This suggests evolution of individual gene family lines of descent by several duplication/specialization events. Using quantitative phylogenetic analyses (RASA, or relative apparent synapomorphy analysis), we demonstrate that comparison of complete MutS protein sequences, rather than highly conserved C-terminal domains only, maximizes information about evolutionary relationships. We identify a novel, highly conserved middle domain, as well as clearly delineate an N-terminal domain, previously implicated in mismatch recognition, that shows family-specific patterns of aromatic and charged amino acids. Our final analysis, in contrast to previous analyses of MutS-like sequences, yields a stable phylogenetic tree consistent with the known biochemical functions of MutS/MSH proteins, that now assigns all known eukaryotic MSH proteins to a monophyletic group, whose branches correspond to the respective specialized gene families. The rooted phylogenetic tree suggests their derivation from a mitochondrial MSH1-like protein, itself the descendent of the MutS of a symbiont in a primitive eukaryotic precursor.

Specific binding of human MSH2.MSH6 mismatch-repair protein heterodimers to DNA incorporating thymine- or uracil-containing UV light photoproducts opposite mismatched bases.

Previous studies have demonstrated recognition of DNA-containing UV light photoproducts by bacterial (Feng, W.-Y., Lee, E., and Hays, J. B. (1991) Genetics 129, 1007-1020) and human (Mu, D., Tursun, M., Duckett, D. R., Drummond, J. T., Modrich, P., and Sancar, A. (1997) Mol. Cell. Biol. 17, 760-769) long-patch mismatch-repair systems. Mismatch repair directed specifically against incorrect bases inserted during semi-conservative DNA replication might efficiently antagonize UV mutagenesis. To test this hypothesis, DNA 51-mers containing site-specific T-T cis-syn-cyclobutane pyrimidine-dimers or T-T pyrimidine-(6-4')pyrimidinone photoproducts, with all four possible bases opposite the respective 3'-thymines in the photoproducts, were analyzed for the ability to compete with radiolabeled (T/G)-mismatched DNA for binding by highly purified human MSH2.MSH6 heterodimer protein (hMutSalpha). Both (cyclobutane-dimer)/AG and ((6-4)photoproduct)/AG mismatches competed about as well as non-photoproduct T/T mismatches. The two respective pairs of photoproduct/(A(T or C)) mismatches also showed higher hMutSalpha affinity than photoproduct/AA "matches"; the apparent affinity of hMutSalpha for the ((6-4)photoproduct)/AA-"matched" substrate was actually less than that for TT/AA homoduplexes. Surprisingly, although hMutSalpha affinities for both non-photoproduct UU/GG double mismatches and for (uracil-cyclobutane-dimer)/AG single mismatches were high, affinity for the (uracil-cyclobutane-dimer)/GG mismatch was quite low. Equilibrium binding of hMutSalpha to DNA containing (photoproduct/base) mismatches and to (T/G)-mismatched DNA was reduced similarly by ATP (in the absence of magnesium).

DNA mismatch repair in plants. An Arabidopsis thaliana gene that predicts a protein belonging to the MSH2 subfamily of eukaryotic MutS homologs.

Sets of degenerate oligomers corresponding to highly conserved domains of MutS-homolog (MSH) mismatch-repair proteins primed polymerase chain reaction amplification of two Arabidopsis thaliana DNA fragments that are homologous to eukaryotic MSH-like genes. Phylogenetic analysis places one complete gene, designated atMSH2, in the evolutionarily distinct MSH2 subfamily.

An Arabidopsis photolyase mutant is hypersensitive to ultraviolet-B radiation.

Photolyases are DNA repair enzymes that use energy from blue light to repair pyrimidine dimers. We report the isolation of an Arabidopsis thaliana mutant (uvr2-1) that is defective in photorepair of cyclobutylpyrimidine dimers (CPDs). Whereas uvr2-1 is indistinguishable from wild type in the absence of UV light, low UV-B levels inhibit growth and cause leaf necrosis. uvr2-1 is more sensitive to UV-B than wild type when placed under white light after UV-B treatment. In contrast, recovery in darkness or in light lacking photoreactivating blue light results in equal injury in uvr2-1 and wild type. The uvr2-1 mutant is unable to remove CPDs in vivo, and plant extracts lack detectable photolyase activity. This recessive mutation segregates as a single gene located near the top of chromosome 1, and is a structural gene mutation in the type II CPD photolyase PHR1. This mutant provides evidence that CPD photolyase is required for plant survival in the presence of UV-B light.

PHH1, a novel gene from Arabidopsis thaliana that encodes a protein similar to plant blue-light photoreceptors and microbial photolyases.

A cDNA from Arabidopsis thaliana similar to microbial photolyase genes, and designated AT-PHH1, was isolated using a photolyase-like cDNA from Sinapsis alba (SA-PHR1) as a probe. Multiple isolations yielded only PHH1 cDNAs, and a few blue-light-receptor CRY1 (HY4) cDNAs (also similar to microbial photolyase genes), suggesting the absence of any other highly similar Arabidopsis genes. The AT-PHH1 and SA-PHR1 cDNA sequences predict 89% identity at the protein level, except for an AT-PHH1 C-terminal extension (111 amino acids), also not seen in microbial photolyases. AT-PHH1 and CRY1 show less similarity (54% p4erein identity), including respective C-terminal extensions that are themselves mostly dissimilar. Analysis of fifteen AT-PHH1 genomic isolates reveals a single gene, with three introns in the coding sequence and one in the 5'-untranslated leader. Full-length AT-PHH1, and both AT-PHH1 and AT-PHH1 delta C-513 (truncated to be approximately the size of microbial photolyase genes) cDNAs, were overexpressed, respectively, in yeast and Escherichia coli mutants hypersensitive to ultraviolet light. The absence of significant effects on resistance suggests either that any putative AT-PHH1 DNA repair activity requires cofactors/chromophores not present in yeast or E. coli, or that AT-PHH1 encodes a blue-light/ultraviolet-A receptor rather than a DNA repair protein.

Developmental responses of amphibians to solar and artificial UVB sources: a comparative study.

Many amphibian species, in widely scattered locations, currently show population declines and/or reductions in range, but other amphibian species show no such declines. There is no known single cause for these declines. Differential sensitivity to UVB radiation among species might be one contributing factor. We have focused on amphibian eggs, potentially the most UVB-sensitive stage, and compared their resistance to UVB components of sunlight with their levels of photolyase, typically the most important enzyme for repair of the major UV photoproducts in DNA, cyclobutane pyrimidine dimers. Photolyase varied 100-fold among eggs/oocytes of 10 species. Among three species-Hyla regilla, Rana cascadae, and Bufo boreas-for which resistance of eggs to solar UVB irradiance in their natural locations was measured, hatching success correlated strongly with photolyase. Two additional species, Rana aurora and Ambystoma gracile, now show similar correlations. Among the low-egg-photolyase species, R. cascadae and B. boreas are showing declines, and the status of A. gracile is not known. Of the two high-photolyase species, populations of H. regilla remain robust, but populations of R. aurora are showing declines. To determine whether levels of photolyase or other repair activities are affected by solar exposures during amphibian development, we have initiated an extended study of H. regilla and R. cascadae, and of Xenopus laevis, laboratory-reared specimens of which previously showed very low photolyase levels. Hyla regilla and R. cascadae tadpoles are being reared to maturity in laboratories supplemented with modest levels of UV light or light filtered to remove UVB wavelengths. Young X. laevis females are being reared indoors and outdoors. Initial observations reveal severe effects of both UVA and UVB light on H. regilla and R. cascadae tadpoles and metamorphs, including developmental abnormalities and high mortalities. Assays of photolyase levels in the skins of young animals roughly parallel previous egg/oocyte photolyase measurements for all three species.

DNA structures generated during recombination initiated by mismatch repair of UV-irradiated nonreplicating phage DNA in Escherichia coli: requirements for helicase, exonucleases, and RecF and RecBCD functions.

During infection of homoimmune Escherichia coli lysogens ("repressed infections"), undamaged nonreplicating lambda phage DNA circles undergo very little recombination. Prior UV irradiation of phages dramatically elevates recombinant frequencies, even in bacteria deficient in UvrABC-mediated excision repair. We previously reported that 80-90% of this UvrABC-independent recombination required MutHLS function and unmethylated d(GATC) sites, two hallmarks of methyl-directed mismatch repair. We now find that deficiencies in other mismatch-repair activities--UvrD helicase, exonuclease I, exonuclease VII, RecJ exonuclease--drastically reduce recombination. These effects of exonuclease deficiencies on recombination are greater than previously observed effects on mispair-provoked excision in vitro. This suggests that the exonucleases also play other roles in generation and processing of recombinagenic DNA structures. Even though dsDNA breaks are thought to be highly recombinagenic, 60% of intracellular UV-irradiated phage DNA extracted from bacteria in which recombination is low--UvrD-, ExoI-, ExoVII-, or Rec(J-)--displays (near-)blunt-ended dsDNA ends (RecBCD-sensitive when deproteinized). In contrast, only bacteria showing high recombination (Mut+ UvrD+ Exo+) generate single-stranded regions in nonreplicating UV-irradiated DNA. Both recF and recB recC mutations strikingly reduce recombination (almost as much as a recF recB recC triple mutation), suggesting critical requirements for both RecF and RecBCD activity. The mismatch repair system may thus process UV-irradiated DNA so as to initiate more than one recombination pathway.

UV repair and resistance to solar UV-B in amphibian eggs: a link to population declines?

The populations of many amphibian species, in widely scattered habitats, appear to be in severe decline; other amphibians show no such declines. There is no known single cause for the declines, but their widespread distribution suggests involvement of global agents--increased UV-B radiation, for example. We addressed the hypothesis that differential sensitivity among species to UV radiation contributes to these population declines. We focused on species-specific differences in the abilities of eggs to repair UV radiation damage to DNA and differential hatching success of embryos exposed to solar radiation at natural oviposition sites. Quantitative comparisons of activities of a key UV-damage-specific repair enzyme, photolyase, among oocytes and eggs from 10 amphibian species were reproducibly characteristic for a given species but varied > 80-fold among the species. Levels of photolyase generally correlated with expected exposure of eggs to sunlight. Among the frog and toad species studied, the highest activity was shown by the Pacific treefrog (Hyla regilla), whose populations are not known to be in decline. The Western toad (Bufo boreas) and the Cascades frog (Rana cascadae), whose populations have declined markedly, showed significantly lower photolyase levels. In field experiments, the hatching success of embryos exposed to UV radiation was significantly greater in H. regilla than in R. cascadae and B. boreas. Moreover, in R. cascadae and B. boreas, hatching success was greater in regimes shielded from UV radiation compared with regimes that allowed UV radiation. These observations are thus consistent with the UV-sensitivity hypothesis.

Selection of Arabidopsis cDNAs that partially correct phenotypes of Escherichia coli DNA-damage-sensitive mutants and analysis of two plant cDNAs that appear to express UV-specific dark repair activities.

To resist terrestrial UV radiation, plants employ DNA-damage-repair/toleration (DRT) activities, as well as shielding mechanisms. Little is known about the structure and regulation of plant DRT genes. We isolated DRT cDNAs from Arabidopsis thaliana, by selecting for complementation of Escherichia coli mutants lacking all bacterial defenses against UV-light damage to DNA. These mutants are phenotypically deficient in recombinational and mutagenic toleration (RecA-), excision repair (Uvr-) and photoreactivation (Phr-). Among 840 survivors of heavily UV-irradiated (10(-7) survival) mutants harboring plasmids derived from an Arabidopsis cDNA library in the vector lambda YES, we identified four unique plant cDNAs, designated DRT100, DRT101, DRT102, and DRT103. Drt101 and Drt102 activity were specific for UV-light damage, and complemented both UvrB- and UvrC- phenotypes in the dark. Apparent Uvr- correction efficiencies were 1 to 40% for Drt101, and 0.2 to 15% for Drt102, depending on the UV fluence. Drt101 and Drt102 showed no extensive amino-acid homology with any known DNA-repair proteins. Drt100 appeared to correct RecA-, rather than Uvr-, phenotypes. Although the light dependence of Drt103 activity was consistent with its identification as a photoreactivating enzyme, its predicted amino-acid sequence did not resemble known photolyase sequences. The N-terminal coding sequence of Drt101 suggests that it is targeted to chloroplasts, as reported for Drt100. These cDNAs afforded only modest increases in survival during the original selection procedure. The fact that they were readily isolated nevertheless suggests that selections may be made powerful enough to overcome barriers to expression and function in bacteria, at least for cDNAs of reasonable abundance.

Two cDNAs from the plant Arabidopsis thaliana that partially restore recombination proficiency and DNA-damage resistance to E. coli mutants lacking recombination-intermediate-resolution activities.

Escherichia coli ruvC recG mutants lack RuvC endonuclease, which resolves crossed-strand joint molecules (Holliday junctions) formed during homologous recombination into recombinant products, and an activity (RecG) thought to partially replace RuvC. They are therefore highly deficient in homologous recombination, and sensitive to UV light and chemical DNA-damaging agents, presumably because of inability to tolerate unrepaired DNA damage by recombinational mechanisms (Lloyd, R.G. (1991) J. Bacteriol. 173:5414-5418). We transformed these mutants with plasmids expressing cDNAs from the plant Arabidopsis thaliana. Selection for bacteria with increased resistance to methylmethanesulfonate yielded two cDNAs, designated DRT111 and DRT112 (DNA-damage-repair/toleration). Expression of these plant cDNAs, especially DRT111, restored conjugal recombination proficiencies in ruvC and ruvC recG mutants to nearly wild-type levels. Both plant cDNAs significantly increased resistance of both mutants to UV light and several chemical DNA-damaging agents, but did not fully correct the mutant phenotypes. Drt111 activity, but not Drt112, also increased, to nearly wild-type levels, resistance of recG single mutants to UV plus mitomycin C. The predicted Drt111 and Drt112 polypeptides, 383 and 167 amino acids respectively, show no similarity with one another or with prokaryotic Holliday resolvases. Both appear chloroplast targeted; Drt112 is highly homologous to Arabidopsis plastocyanin. DRT111 and DRT112 probes hybridize only to DNA from closely related plants.

A plant cDNA that partially complements Escherichia coli recA mutations predicts a polypeptide not strongly homologous to RecA proteins.

A plant (Arabidopsis thaliana) cDNA previously selected for its ability to partially complement the UV sensitivity of Escherichia coli RecA-UvrC-Phr- mutants and designated DRT100 (DNA-damage repair/toleration) was subcloned into a high-copy-number plasmid and expressed via a bacterial promotor. It increased resistance of RecA-UvrB-Phr- bacteria to mitomycin C and methyl methanesulfonate as well as to UV light. This lack of specificity, and its ability to increase resistance in both UvrB- and UvrC- mutants, suggested that Drt100 activity might be complementing RecA- phenotypes. DRT100 partially complemented three RecA- phenotypes thought to reflect deficiencies in homologous recombination--namely, inability to plate lambda red-gam- phages and P1 phages and to recombinationally integrate donor DNA during conjugal crosses--but did not complement inability to induce E. coli SOS functions. The 395-amino acid DRT100 open reading frame encodes an apparent N-terminal chloroplast transit peptide and a putative 322-residue mature protein with a conserved nucleotide binding motif, but otherwise little global homology with bacterial RecA proteins. There are several tandemly repeated leucine-rich motifs. DNA from two closely related plants, but not from maize, hybridized strongly to a DRT100 cDNA probe.