DNA cytosine C5 methyltransferase Dnmt1: catalysis-dependent release of allosteric inhibition

We followed the cytosine C(5) exchange reaction with Dnmt1 to characterize its preference for different DNA substrates, its allosteric regulation, and to provide a basis for comparison with the bacterial enzymes. We determined that the methyl transfer is rate-limiting, and steps up to and including the cysteine-cytosine covalent intermediate are in rapid equilibrium. Changes in these rapid equilibrium steps account for many of the previously described features of Dnmt1 catalysis and specificity including faster reactions with premethylated DNA versus unmethylated DNA, faster reactions with DNA in which guanine is replaced with inosine [poly(dC-dG) vs poly(dI-dC)], and 10-100-fold slower catalytic rates with Dnmt1 relative to the bacterial enzyme M.HhaI. Dnmt1 interactions with the guanine within the CpG recognition site can prevent the premature release of the target base and solvent access to the active site that could lead to mutagenic deamination. Our results suggest that the beta-elimination step following methyl transfer is not mediated by free solvent. Dnmt1 shows a kinetic lag in product formation and allosteric inhibition with unmethylated DNA that is not observed with premethylated DNA. Thus, we suggest the enzyme undergoes a slow relief from allosteric inhibition upon initiation of catalysis on unmethylated DNA. Notably, this relief from allosteric inhibition is not caused by self-activation through the initial methylation reaction, as the same effect is observed during the cytosine C(5) exchange reaction in the absence of AdoMet. We describe limitations in the Michaelis-Menten kinetic analysis of Dnmt1 and suggest alternative approaches.     

Kinetic mechanism of cytosine DNA methyltransferase MspI.

A kinetic analysis of MspI DNA methyltransferase (M.MspI) is presented. The enzyme catalyzes methylation of lambda-DNA, a 50-kilobase pair linear molecule with multiple M.MspI-specific sites, with a specificity constant (kcat/KM) of 0.9 x 10(8) M-1 s-1. But the values of the specificity constants for the smaller DNA substrates (121 and 1459 base pairs (bp)) with single methylation target or with multiple targets (sonicated lambda-DNA) were less by an order of magnitude. Product inhibition of the M.MspI-catalyzed methylation reaction by methylated DNA is competitive with respect to DNA and noncompetitive with respect to S-adenosylmethionine (AdoMet). The S-adenosylhomocysteine inhibition of the methylation reaction is competitive with respect to AdoMet and uncompetitive with respect to DNA. The presteady state kinetic analysis showed a burst of product formation when AdoMet was added to the enzyme preincubated with the substrate DNA. The burst is followed by a constant rate of product formation (0.06 mol per mol of enzyme s-1) which is similar to catalytic constants (kcat = approximately 0.056 s-1) measured under steady state conditions. The isotope exchange in chasing the labeled methyltransferase-DNA complex with unlabeled DNA and AdoMet leads to a reduced burst as compared with the one involving chase with labeled DNA and AdoMet. The enzyme is capable of exchanging tritium at C-5 of target cytosine in the substrate DNA in the absence of cofactor AdoMet. The kinetic data are consistent with an ordered Bi Bi mechanism for the M.MspI-catalyzed DNA methylation where DNA binds first.     

Flowering of Arabidopsis cop1 mutants in darkness

To elucidate the role of the COP1 gene in flowering, we analyzed flowering of cop1 mutant lines in darkness. When grown in the presence of 1% (w/v) sucrose, the cop1-6 mutant flowered in darkness, but cop1-1 and cop1-4 did not. However, cop1-1 and cop1-4 flowered in darkness when grown in the presence of 5% (w/v) sucrose. Therefore, the COP1 gene represses not only photomorphogenesis in seedlings but also flowering in darkness. Comparison of mRNAs levels of floral identity genes in cop1-6 and wild-type plants grown in darkness revealed increased mRNA levels of genes that act downstream of CO and reduced FLC mRNA level in cop1-6. Double mutants of cop1-6 and each of the late-flowering mutations cry2-1, gi-2, co-1, and ld-1 flowered in darkness. All of the double mutants except cry2-1 cop1-6 flowered later than cop1-6, demonstrating that cop1-6 is epistatic to cry2-1 for early flowering. The ld-1 cop1-6 double mutant flowered much earlier than the ld-1 mutant. The delay in flowering in the double mutants was not strongly influenced by the light conditions, whereas that of the gi-2 cop1-6 double mutant was reduced in darkness.     

Cytokinesis-defective mutants of Arabidopsis

We have identified mutations in six previously uncharacterized genes of Arabidopsis, named club, bublina, massue, rod, bloated, and bims, that are required for cytokinesis. The mutants are seedling lethal, have morphological abnormalities, and are characterized by cell wall stubs, gapped walls, and multinucleate cells. In these and other respects, the new mutants are phenotypically similar to knolle, keule, hinkel, and pleiade mutants. The mutants display a gradient of stomatal phenotypes, correlating roughly with the severity of their cytokinesis defect. Similarly, the extent to which the different mutant lines were capable of growing in tissue culture correlated well with the severity of the cytokinesis defect. Phenotypic analysis of the novel and previously characterized loci indicated that the secondary consequences of a primary defect in cytokinesis include anomalies in body organization, organ number, and cellular differentiation, as well as organ fusions and perturbations of the nuclear cycle. Two of the 10 loci are required for both cytokinesis and root hair morphogenesis. The results have implications for the identification of novel cytokinesis genes and highlight the mechanistic similarity between cytokinesis and root hair morphogenesis, two processes that result in a rapid deposition of new cell walls via polarized secretion.     

Embryonic mutants of Arabidopsis thaliana

Genetic analysis of plant em-bryogenesis has been approached in part through the isolation and characterization of recessive embryonic mutants. The most extensive studies have dealt with maize and Arabidopsis. The high frequency of mutants defective in plant embryogenesis is consistent with the presence of many target genes with essential functions at this stage of the life cycle. Some mutants are likely to be defective in genes with general housekeeping functions. Others should facilitate the identification of genes with a more direct role in the regulation of morphogesis. Over 300 embryonic mutants of Arabidopsis isolated following chemical mutagenesis and T-DNA insertional mutagenesis are currently being analyzed. This collection includes embryonic le-thals, defectives, and pattern mutants. Developmental abnormalities include the presence of fused cotyledons, twin embryos, abnormally large suspensors, distorted epidermal layers, single cotyledons, enlarged shoot apices, pattern deletions and duplications, embryos with altered patterns of symmetry, bloated embryos with giant vacuolated cells, reduced hypocotyls that fail to produce roots, and embryos that protrude through the seed coat late in maturation. This review describes the isolation and characterization of embryonic mutants of Arabidopsis and their potential application to plant biology. © 1992 Wiley-Liss, Inc.     

Floral transition mutants in Arabidopsis

An inventory of genetic differences in flowering time in Arabidopsis is presented and discussed. Many genes influence the transition to flowering in a quantitative way. Two groups of mutants and natural variants can be distinguished: those that are responsive to environmental factors and those that are less responsive or unresponsive. It is possible that all late/early-flowering mutants isolated to date carry a mutation with an effect, either promotive or repressive, on a floral repressor. The interaction between light perception and flowering has been studied by analysis of phytochrome- and cryptochrome-deficient mutants, which showed that phyA and probably also cryptochrome have a promotive role in flowering, whereas phyB and other stable phytochromes have an inhibitory role. A circadian rhythm is important in establishing daylength sensitivity, as was shown by the phenotype of the elf 3 mutants.     

The gene for domains rearranged methyltransferase (DRM2) in Arabidopsis thaliana plants is methylated at both cytosine and adenine residues

The methylation patterns of cytosine and adenine residues in the Arabidopsis thaliana gene for domains rearranged methyltransferase (DRM2) were studied in wild-type and several transgene plant lines containing antisense fragments of the cytosine DNA-methyltransferase gene METI under the control of copper-inducible promoters. It was shown that the promoter region of the DRM2 gene is mostly unmethylated at the internal cytosine residue in CCGG sites whereas the 3'-end proximal part of the gene coding region is highly methylated. The DRM2 gene was found to be also methylated at adenine residues in some GATC sequences. Cytosine methylation in CCGG sites and adenine methylation in GATC sites in the DRM2 gene are variable between wild-type and different transgenic plants. The induction of antisense METI constructs with copper ions in transgene plants in most cases leads to further alterations in the DRM2 gene methylation patterns.     

Cesium-insensitive mutants of Arabidopsis thaliana

The molecular analysis of solute transport across the plasma membrane in animals and microorganisms has been aided by the analysis of well-defined transport mutants. To obtain mutant plants with genetic defects in cation transport, the inhibitory effect of monovalent cations (Li⁺, Na⁺, Rb⁺, and Cs⁺) on Arabidopsis thaliana seed germination was tested. Cesium was unique in that at low concentration it strongly inhibited seedling development. In this report it is demonstrated that cesium is a competitive inhibitor for potassium transport in A. thaliana and its toxicity is closely tied to the level of potassium supplied. Conditions were obtained to maximize the cesium-sensitivity for seed germination in a large population, and selection for resistance using M₂ seeds derived from ethyl methane sulfonate (EMS)-treated plants yielded several dozen resistant plants. Seeds derived from these plants yielded cesium-insensitive mutant lines with heritable changes in energy-dependent potassium uptake. In progeny from a backcross to wild-type plants, at least one of the lines showed the segregation ratio expected for a single-gene recessive mutation and an RFLP analysis mapped the mutant locus to the top of chromosome 4.     

Alteration of auxin polar transport in the Arabidopsis ifl1 mutants

The INTERFASCICULAR FIBERLESS/REVOLUTA (IFL1/REV) gene is essential for the normal differentiation of interfascicular fibers and secondary xylem in the inflorescence stems of Arabidopsis. It has been proposed that IFL1/REV influences auxin polar flow or the transduction of auxin signal, which is required for fiber and vascular differentiation. Assay of auxin polar transport showed that the ifl1 mutations dramatically reduced auxin polar flow along the inflorescence stems and in the hypocotyls. The null mutant allele ifl1-2 was accompanied by a significant decrease in the expression level of two putative auxin efflux carriers. The ifl1 mutants remained sensitive to auxin and an auxin transport inhibitor. The ifl1-2 mutant exhibited visible phenotypes associated with defects in auxin polar transport such as pin-like inflorescence, reduced numbers of cauline branches, reduced numbers of secondary rosette inflorescence, and dark green leaves with delayed senescence. The visible phenotypes displayed by the ifl1 mutants could be mimicked by treatment of wild-type plants with an auxin polar transport inhibitor. In addition, the auxin polar transport inhibitor altered the normal differentiation of interfascicular fibers in the inflorescence stems of wild-type Arabidopsis. Taken together, these results suggest a correlation between the reduced auxin polar transport and the alteration of cell differentiation and morphology in the ifl1 mutants.     

Genetic and phenotypic characterization of cop1 mutants of Arabidopsis thaliana

Ten Arabidopsis lines that carry recessive mutations in the cop1 (constitutively photomorphogenic) locus have been isolated. These lines define at least four different alleles. All of the mutant lines produce dark-grown seedlings that mimic wild-type seedlings grown in the light. The phenotype of the dark-grown mutant seedlings includes: short hypocotyls, open and enlarged cotyledons, accumulation of anthocyanin, cell-type differentiation and chloroplast-like plastid differentiation in cotyledons. Moreover, in more prolonged dark-growth periods the mutants exhibit true leaf development that parallels that in light-grown siblings. The four mutant alleles represent two types of mutations: three alleles (cop 1-1, cop 1-2, and cop 1-3) have severely affected phenotypes whereas one allele (cop 1-4) has a less severe phenotype. Compared to the severe alleles, the cop 1-4 mutant has slightly longer hypocotyls in dark-grown seedlings and does not accumulate abnormal levels of anthocyanin. The cop1–1/cop1-4 hybrid seedlings are intermediate in many physiological properties under both dark- and light-growth conditions, relative to the two parents. These results may suggest that the extent of residual cop1 gene activity in the mutants dictates the degree to which the aberrant plant phenotype is expressed. Analysis of plants carrying both cop1 and hy, a mutation that results in a deficiency of active phyto-chrome, suggests that the cop1 gene product acts downstream of phytochrome. The differentiation of chloroplasts in the roots of light-grown cop1 plants but not in wild-type plants suggests that the wild-type cop1 gene product also normally plays a role in suppressing chloroplast development in the roots of light-grown plants. To aid the eventual molecular cloning of the cop1 locus, its chromosomal location has been mapped and a molecular marker that is located about 1 centimorgan away from the cop1 locus obtained.     

Growth and development of the axr1 mutants of Arabidopsis.

We have recovered eight new auxin-resistant lines of Arabidopsis that carry mutations in the AXR1 gene. These eight lines, together with the 12 lines described in a previous report, define at least five different axr1 alleles. All of the mutant lines have a similar phenotype. Defects include decreases in plant height, root gravitropism, hypocotyl elongation, and fertility. Mutant line axr1-3 is less resistant to auxin than the other mutant lines and has less severe morphological abnormalities. This correlation suggests that the morphological defects are a consequence of a defect in auxin action. To determine whether the altered morphology of mutant plants is associated with changes in cell size or tissue organization, tissue sections were examined using scanning electron microscopy. No clear differences in cell size were observed between wild-type and mutant tissues. However, the vascular bundles of mutant stems were found to be less well differentiated than those in wild-type stems. The auxin sensitivity of rosette-stage plants was determined by spraying plants with auxin solutions. Mutant rosettes were found to be significantly less sensitive to exogenously applied auxin than wild-type rosettes, indicating that the AXR1 gene functions in aerial portions of the plant. Our studies suggest that the AXR1 gene is required for auxin action in most, if not all, tissues of the plant and plays an important role in plant development. Linkage studies indicate that the gene is located on chromosome 1 approximately 2 centiMorgans from the closest restriction fragment length polymorphism.     

A potent cell-active allosteric inhibitor of murine DNA cytosine C5 methyltransferase

The major DNA cytosine methyltransferase isoform in mouse erythroleukemia cells, Dnmt1, exhibits potent dead-end inhibition with a single-stranded nucleic acid by binding to an allosteric site on the enzyme. The previously reported substrate inhibition with double-stranded substrates also involves binding to an allosteric site. Thus, both forms of inhibition involve ternary enzyme-DNA-DNA complexes. The inhibition potency of the single-stranded nucleic acid is determined by the sequence, length, and most appreciably the presence of a single 5-methylcytosine residue. A single-stranded phosphorothioate derivative inhibits DNA methylation activity in nuclear extracts. Mouse erythroleukemia cells treated with the phosphorothioate inhibitor show a significant decrease in global genomic methylation levels. Inhibitor treatment of human colon cancer cells causes demethylation of the p16 tumor suppressor gene and subsequent p16 re-expression. Allosteric inhibitors of mammalian DNA cytosine methyltransferases, representing a new class of molecules with potential therapeutic applications, may be used to elucidate novel epigenetic mechanisms that control development.