( 6 N)methyl adenine in the nuclear DNA of a eucaryote, Tetrahymena pyriformis.

DNA isolated from macronuclei of the ciliate, Tetrahymena pyriformis, has been found to contain [(6)N]methyl adenine (MeAde); this represents the first clear demonstration of significant amounts of MeAde in the DNA of a eucaryote. The amounts of macronuclear MeAde differed slightly between different strains of Tetrahymena, with approximately 0.65-0.80% of the adenine bases being methylated. The MeAde content of macronuclear DNA did not seem to vary in different physiological states. The level of MeAde in DNA isolated from micronuclei, on the other hand, was quite low (at least tenfold lower than in macronuclear DNA).

Deoxyribonucleic acid methylation and chromatin organization in Tetrahymena thermophila.

Deoxyribonucleic acid (DNA) of the transcriptionally active macronucleus of Tetrahymena thermophila is methylated at the N6 position of adenine to produce methyladenine (MeAde); approximately 1 in every 125 adenine residues (0.8 mol%) is methylated. Transcriptionally inert micronuclear DNA is not methylated (< or = 0.01 mol% MeAde; M. A. Gorovsky, S. Hattman, and G. L. Pleger, J. Cell Biol. 56:697-701, 1973). There is no detectable cytosine methylation in macronuclei in Tetrahymena DNA (< or = 0.01 mol% 5-methylcytosine). MeAde-containing DNA sequences in macronuclei are preferentially digested by both staphylococcal nuclease and pancreatic deoxyribonuclease I. In contrast, there is no preferential release of MeAde during digestion of purified DNA. These results indicate that MeAde residues are predominantly located in "linker DNA" and perhaps have a function in transcription. Pulse-chase studies showed that labeled MeAde remains preferentially in linker DNA during subsequent rounds of DNA replication; i.e., there is little, if any, movement of nucleosomes during chromatin replication. This implies that nucleosomes may be phased with respect to DNA sequence.

Relationship of DNA methylation level to the presence of heterochromatin in mealybugs.

Purified nuclear DNA from two mealybug species was analyzed for its 5-methylcytosine (m5C) content by reversed-phase high-pressure liquid chromatography. We observed that the percent m5C (percentage of cytosines which are methylated) varied between the two species, between males and females of the same species, and between lines with and without supernumerary B chromosomes. This is the first case of a sex-specific difference in overall DNA methylation level. In contrast to a recent report (Deobagkar et al., J. Biosci. [India] 4:513-526, 1982), we found no other modified bases in the DNA. Overall, the percent m5C in Pseudococcus obscurus was two to three times higher than in Pseudococcus calceolariae. In both species, the percent m5C in males was higher than in females, although only in P. calceolariae was the difference statistically significant (0.68 +/- 0.02 versus 0.44 +/- 0.04). The high m5C content in males was correlated with the presence of a paternally derived, genetically inactive set of chromosomes which is facultatively heterochromatic. The presence of constitutive heterochromatin, however, was associated with a lower m5C content. Thus, for example, the percent m5C in females of a P. obscurus line with heterochromatic B chromosomes (1.09 +/- 0.04) was significantly lower than that of a related line lacking such chromosomes (1.26 +/- 0.06). Our findings are discussed with respect to the possible relationship between DNA methylation and heterochromatization.

5-Methylcytosine is not detectable in Saccharomyces cerevisiae DNA.

We examined the DNA of Saccharomyces cerevisiae by both HpaII-MspI restriction enzyme digestion and high-performance liquid chromatography analysis for the possible presence of 5-methylcytosine. Both of these methods failed to detect cytosine methylation within this yeast DNA; i.e., there is less than 1 5-methylcytosine per 3,100 to 6,000 cytosine residues.

Methylation by a mutant T2 DNA [N(6)-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage.

Properties of a mutant bacteriophage T2 DNA [N:(6)-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose.

Pre-steady state kinetics of bacteriophage T4 dam DNA-[N(6)-adenine] methyltransferase: interaction with native (GATC) or modified sites.

The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S:-adenosyl-L-methionine (AdoMet) to the N(6)-position of adenine [generating N(6)-methyladenine and S:-adenosyl-L-homocysteine (AdoHcy)]. Pre-steady state kinetic analysis revealed that the methylation rate constant k(meth) for unmethylated and hemimethylated substrates (0.56 and 0.47 s(-1), respectively) was at least 20-fold larger than the overall reaction rate constant k(cat) (0.023 s(-1)). This indicates that the release of products is the rate-limiting step in the reaction. Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleoside flipping does not limit k(meth). Preformed T4 Dam MTase-DNA complexes are less efficient than preformed T4 Dam MTase-AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.

A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase.

The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix. Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an (2)A/(2)A double-substituted duplex. Since the (2)A/(m)A duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se. We propose that T4 Dam alone randomly binds to the asymmetric (2)A/A and (2)A/(m)A duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor. The results of pre-steady-state methylation kinetics are consistent with this model.

Unusual transcriptional and translational regulation of the bacteriophage Mu mom operon.

The bacteriophage Mu mom gene encodes a novel DNA modification that protects the viral genome against a wide variety of restriction endonucleases. Expression of mom is subject to a series of unusual regulatory controls. Transcription requires the action of a phage-encoded protein, C, which binds (probably as a dimer) the mom promoter from -33 to -52 (with respect to the transcription start site) in two adjacent DNA major grooves on one face of the helix. No apparent direct interaction between C and the host RNA polymerase (RNAP) is evident; however, C binding alters mom DNA conformation. In the absence of C, RNAP binds the mom promoter at a site that results in transcription in a direction away from the mom gene. The function of this transcription is unknown. An additional layer of transcriptional regulation complexity is due to the fact that the host Dam DNA-(N6-adenine)methyltransferase is required. Dam methylation of three closely spaced upstream GATC sequences is necessary to prevent binding by the host protein, OxyR, which acts as a repressor. Repression is not mediated by inhibition of C binding, but rather through interference with C-mediated recruitment of RNAP to the correct site. Translation of mom is regulated by the phage Com protein. Com is only 62 amino acids long and contains a zinc finger-like structure (coordinated by four cysteine residues) in the amino terminal domain. Com binds mom mRNA 5' to the mom open reading frame, whose translation start signals are contained in a stem-loop translation-inhibition-structure. Com binding to its target site (5' to and adjacent to the translation-inhibition-structure) results in a stable change in RNA secondary structure that exposes the translation start signals.

Bidirectional transcription in the mom promoter region of bacteriophage Mu.

Transcription of the Mu mom operon requires activation by the phage gene product, C, a site-specific DNA binding protein. Previous in vivo and in vitro footprinting studies showed that Escherichia coli RNA polymerase (Esigma70=RNAP) bound the wild-type (wt) mom promoter (Pmom) region in the absence of C; this site, now designated momP2 (-11 to -64), is slightly upstream of, but overlapping with, momP1 (+16 to -49), the functional binding site for mom operon (rightward) transcription. The location/distribution of KMnO4-sensitive sites on the two DNA strands suggested that RNAP bound at momP2 was in an open-complex, but that transcription was in the opposite direction. Here, we used both runoff transcription and reverse transcriptase-primer extension sequencing to provide direct evidence that in the absence of C protein, RNAP carries out leftward transcription from momP2 both in vitro and in vivo. In addition, the 5' ends of these transcripts were mapped to the same upstream initiation site, -58G, relative to the initiation site of C-activated rightward transcription. We also present evidence that leftward transcription from momP2 requires RNAP recognition of an UP-element by the carboxyl-terminal domain of the alpha subunit.

Interaction of the bacteriophage Mu transcriptional activator protein, C, with its target site in the mom promoter.

The bacteriophage Mu C gene encodes a 16.5 kDa site-specific DNA binding protein that is a transcriptional activator of the four "late" promoters, Pmom, Plys, PI and PP. A symmetrical consensus C recognition sequence, TTAT[N5-6]ATAA, containing an inverted tetrad repeat separated by a spacer of five to six G+C-rich nucleotides, has been proposed. To investigate this, we used oligonucleotide mutagenesis to introduce random substitutions within and flanking the proposed C-target region; each variant site was tested for C recognition by an in vivo functional transactivation assay. We observed that all single mutations, in either tetrad, reduced C activation. Although two out of ten substitutions within the spacer reduced activation, the spacer region does not appear to make specific contact with C. We also used in vitro chemical-protection and -interference to study C contacts with Pmom. The results indicate that C contacts Pmom DNA on only one face of the helix through interactions within two adjacent major grooves; this conclusion was supported by gel shift analyses using synthetic oligonucleotide duplexes containing I.C or other base-pair substitutions. Evidence is also presented that C-Pmom contacts are asymmetrical, and that they extend two nucleotides 3' to the promoter-proximal tetrad. We also show that C binding induces a deformation, possibly a bend, in Pmom DNA.

The zinc coordination site of the bacteriophage Mu translational activator protein, Com.

The bacteriophage Mu Com protein is a small "zinc finger-like" protein that binds a specific site in com-mom operon mRNA and activates translation of the mom open-reading-frame. Com contains six cysteine and five histidine residues that have the potential to form several alternative zinc-finger-like motifs. We have used oligonucleotide site-directed mutagenesis to individually alter each of these amino acids (Cys to Ser, and His to Asn or Gln) and tested the various forms of Com for their ability to function in vivo. We observed that mutation of any one of the four N-terminal cysteine residues (Cys-6, 9, 26 or 29) resulted in loss of Com activity. The Com protein requires zinc in order to fold into its functional tertiary structure, as demonstrated by characteristic 1H nuclear magnetic resonance (NMR) chemical shifts. 1H chemical shifts revert to random coil values in the presence of the metal chelator EDTA. The metal-binding specificity and thermal stability of Com also has been investigated using 1H NMR. We report the use of 113Cd NMR, 1H-113Cd heteronuclear spin-echo difference spectroscopy HSED and Zn extended X-ray absorption fine structure spectroscopy EXAFS to determine the zinc/protein stoichiometry as 1:1 and the ligand environment as tetrathiolate. Comparative NMR spectra of Com mutants C6S and C39S suggest position 6 is involved in zinc coordination, while position 39 is not metal-liganded. These studies indicate that the metal coordination, site of Com is a four-cysteine complex, involving residues 6, 9, 26 and 29.

The Escherichia coli MutH protein is not the repressor of the bacteriophage Mu mom operon.

Expression of the bacteriophage Mu mom-operon is under tight regulatory control. One of the factors required for transcription of the operon is the host Escherichia coli Dam activity. It was proposed that DNA methylation by this enzyme prevents the binding of a cellular repressor to an operator site containing three 5'-GATC-3' sequences, the known target site of Dam methylation. Support for this model came from the observation of others that the requirement for Dam was almost completely suppressed in a mutH-lysA deletion mutant, suggesting that the MutH protein is the postulated transcriptional repressor. In this communication, however, I show that the Dam requirement is not effectively relieved in this deletion mutant; therefore, the MutH protein alone is not the mom repressor.

S1 nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression.

The mom gene of bacteriophage Mu encodes a DNA modification function. Expression of this modification requires the host Escherichia coli Dam (DNA-adenine methylase) function and the transacting phage Mu Dad function. The mom gene was subcloned into a variety of sites on plasmid pBR322. Insertions were made into the HincII and PvuI sites within the amp gene and into the ClaI site of the tet gene promoter. The only clones found were those in which the orientation of the mom gene prevents its transcription from the vector promoter(s), suggesting that constitutive expression of mom from a foreign promoter can occur independently of Dad function but is lethal for the cell. Employing S1 nuclease mapping, we have identified two Mu mRNA transcripts: (1) the gin transcript extends into the gin-mon intercistronic divide and terminates downstream from the BclI site; and (2) the mom transcript appears to initiate about 74 bp upstream from the BclI site, 12 bp downstream from a promoter-like sequence. Production of the mom transcript is dependent on the host Dam activity and on Dad transactivation. In contrast, the gin transcript is produced independently of Dam and Dad functions; the gin transcript may extend into the mom gene, but it appears to be either degraded at the 3' end or differentially terminated. We propose that regulation of mom gene transcription involves both positive and negative regulatory proteins, and that binding of the Dad protein (a "late" Mu protein) is required for transcription initiation by the host RNA polymerase. However, Dad protein action may be inhibited by prior binding of a repressor to the mom operator, located farther upstream. We propose that this repressor (encoded by a phage or host gene) binds to the operator only when there is no active Dam enzyme present, i.e., when there is no methylation of (or methylase binding to) the G-A-T-C sites within the mom operator.

Molecular cloning of a functional dam+ gene coding for phage T4 DNA adenine methylase.

Phages T2 and T4 induce synthesis of a DNA-adenine methylase which is coded for by a phage gene, dam+. These enzymes methylate adenine residues in specific sequences which include G-A-T-C, the methylation site of the host Escherichia coli dam+ methylase. Methylation of G-A-T-C to G-m6A-T-C protects the site against cleavage by the MboI restriction nuclease. We have taken advantage of this property to enrich and screen for transformants which contain a cloned, functional T4 dam+ gene. These recombinant molecules consist of a 1.85-kb HindIII fragment inserted into the plasmid pBR322; both orientations of the fragment express the methylase gene, suggesting that transcription is from a T4 promoter. We have tested the 1.85-kb insert for sensitivity to a variety of restriction nucleases and have found single sites for EcoRI, BalI, XbaI, and at least two sites for BstNI (EcoRII). The relative positions of these restriction sites have also been determined. Physical mapping was carried out by Southern blot hybridization with 32P-labeled (nick-translated clone) probe. These experiments showed that the insert corresponds to a HindIII fragment located on the physical map of T4 between positions 16.2 and 18.1 kb from the T4rIIA-rIIB junction. E. coli dam- possesses several phenotypic differences from the wild-type dam+ parent, including an increased sensitivity to 2-aminopurine (2-AP). We found that T4 dam+ clones could relieve dam- cells of their increased sensitivity to 2-AP.

Studies on the function of conserved sequence motifs in the T4 Dam-[N6-adenine] and EcoRII [C5-cytosine] DNA methyltransferases.

We used site-directed oligodeoxyribonucleotide-mediated mutagenesis and kinetic studies with purified wild-type (wt) and mutant proteins to evaluate the role of the conserved sequence motifs in two prokaryotic DNA MTases. We suggest that: (i) the main role of Pro in the M.EcoRII PC-motif is to restrict the conformational freedom of Cys and orient it in a manner essential for catalysis; (ii) in both M.EcoRII and T4 Dam the FXGXG-motif positions AdoMet with respect to the catalytic site; (iii) the DPPY-motif in T4 Dam (region IV) is important for AdoMet-binding and may be part of the binding site; and (iv) the RXNXKXXFXXPFK-motif in T4 Dam (region III) is part of the DNA binding/recognition domain.

Sequence motifs common to the EcoRII restriction endonuclease and the proposed sequence specificity domain of three DNA-[cytosine-C5] methyltransferases.

We have compared the deduced amino acid (aa) sequences of the EcoRII restriction endonuclease (R.EcoRII) and the proposed specificity (target recognition) domains of three DNA-[cytosine-C5] methyltransferases (MTases), M.EcoRII, M.Dcm, and M.SPR, each of which recognizes the same nucleotide sequence, CCWGG (where W is A or T). We have identified a region containing sequence motifs that are partially conserved in the MTases and R.EcoRII. This may be the first example of aa sequence homology between a MTase specificity (target recognition) domain and its cognate restriction endonuclease (ENase). It suggests that this region is important for DNA recognition by R.EcoRII and that the EcoRII ENase and MTase genes may have evolved from a common progenitor.

The bacteriophage Mu com gene appears to specify a translation factor required for mom gene expression.

Expression of the bacteriophage Mu mom gene is subject to a variety of regulatory controls. Both the host Dam DNA-adenine methylase and the phage Mu C protein are required for mom gene transcription. In addition, the Mu com gene product is required for production of the mom protein. Because the com and mom genes overlap on the same mRNA transcript (with com being located proximal to the 5' end), it is likely that Com function is exerted after transcription initiation. To study the role of Com, two segments of Mu were cloned in both orientations (+ and -) into the HindIII site of the galactokinase expression vector, pKG1800; the HindIII site is located between the galK structural gene and its promoter. In (+) plasmids, the Mu DNA inserts were transcribed from the gal promoter in the same orientation as in the phage genome; (-) plasmids had the Mu DNA inserted in the reverse orientation. Each Mu insert contained the same segment of the mom gene from the 3' terminus, but differed in the extent of com gene included at the 5' terminus; one contained a truncated com gene and the other a complete com gene, as well as upstream Mu regulatory sequences. The results are summarized as follows: (1) both (-) plasmids produced only about 10% as much galactokinase activity following fucose induction as the parental vector, pKG1800; (2) plasmid pGTVH(+), with an intact com gene produced about 30% as much galactokinase as pKG1800; (3) plasmid pMTVH(+), with a truncated com gene, produced only about 10% as much enzyme as pKG1800.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation and expression of the bacteriophage mu mom gene: mapping of the transactivation (dad) function to the C region.

Expression of the bacteriophage Mu mom gene is under tight regulatory control. One of the factors required for mom gene expression is the trans-acting function (designated Dad) provided by another Mu gene. To facilitate studies on the signals mediating mom regulation, we have constructed a mom-lacZ fusion plasmid which synthesizes beta-galactosidase only when the Mu Dad transactivating function is provided. lambda pMu phages carrying different segments of the Mu genome have been assayed for their ability to transactivate beta-galactosidase expression by the fusion plasmid. The results of these analyses indicated that the Dad transactivation function is encoded between the leftmost EcoRI site and the lys gene of Mu; this region includes the C gene, which is required for expression of all Mu late genes. Cloning of an approx. 800-bp fragment containing the C gene produced a plasmid which could complement MuC- phages for growth and could transactivate the mom-lacZ fusion plasmid to produce beta-galactosidase. These results suggest that the C gene product mediates the Dad transactivation function.

The DNA [adenine-N6]methyltransferase (Dam) of bacteriophage T4.

A functional bacteriophage T4 dam+ gene, which specifies a DNA [adenine-N6]methyltransferase (Dam), was cloned on a 1.8-kb HindIII fragment [Schlagman and Hattman, Gene 22 (1983) 139-156]. Sequence analysis [Macdonald and Mosig, EMBO J. 3 (1984) 2863-2871] revealed two overlapping in-phase open reading frames (ORFs). The 5' proximal ORF initiates translation at an AUG and encodes a 30-kDa polypeptide, whereas the downstream ORF initiates translation at a GUG and encodes a 26-kDa polypeptide. Analysis of BAL 31 deletions in our original dam+ clone has verified that at least one of these overlapping ORFs, in fact, encodes T4 Dam. To investigate where T4 Dam translation is initiated, we have constructed plasmids in which a tac or lambda PL promoter is placed 5' to either the longer ORF or just the shorter ORF. Only clones which contain a promoter in front of the longer ORF produce active T4 Dam. This indicates that the 26-kDa polypeptide alone cannot be T4 Dam. Additional experiments suggest that only the 30-kDa polypeptide is required for enzyme activity and that the shorter ORF is not translated in plasmid-carrying cells. We also present evidence that T4 Dam is capable of methylating 5'-GATC-3', GATm5C, and GAThmC sequences; non-canonical sites (e.g., GACC) are also methylated, but much less efficiently.

Function of Pro-185 in the ProCys of conserved motif IV in the EcoRII [cytosine-C5]-DNA methyltransferase.

ProCys in the conserved sequence motif IV of [cytosine-C5]-DNA methyltransferases is known to be part of the catalytic site. The Cys residue is directly involved in forming a covalent bond with the C6 of the target cytosine. We have found that substitution of Pro-185 with either Ala or Ser resulted in a reduced rate of methyl group transfer by the EcoRII DNA methyltransferase. In addition, we observed an increase in the Km for substrate S-adenosyl-L-methionine (AdoMet), but a decrease in the Km for substrate DNA. This is reflected in minor changes in kcat/Km for DNA, but in 10- to 100-fold reductions in kcat/Km for AdoMet. This suggests that Pro-185 is important to properly orient the activated cytosine and AdoMet for methyl group transfer by direct interaction with AdoMet and indirectly via the Cys interaction with cytosine.