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.

PCR fragmentation of DNA.

A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.

Effect of base analog substitutions in the specific GATC site on binding and methylation of oligonucleotide duplexes by the bacteriophage T4 Dam DNA-[N6-adenine] methyltransferase.

The interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with 24mer synthetic oligonucleotide duplexes having different purine base substitutions in the palindromic recognition sequence, GATC, was investigated by means of gel shift and methyl transfer assays. The substitutions were introduced in either the upper or lower strand: guanine by 7-deazaguanine (G-->D) or 2-aminopurine (G-->N) and target adenine by purine (A-->P) or 2-aminopurine (A-->N). The effects of each base modification on binding/methylation were approximately equivalent for both strands. G-->D and G-->N substitutions resulted in a sharp decrease in binary complex formation. This suggests that T4 Dam makes hydrogen bonds with either the N7- or O6-keto groups (or both) in forming the complex. In contrast, A-->P and A-->N substitutions were much more tolerant for complex formation. This confirms our earlier observations that the presence of intact 5'-G:C base pairs at both ends of the methylation site is critical, but that base substitutions within the central A:T base pairs show less inhibition of complex formation. Addition of T4 Dam to a complete substrate mixture resulted in a burst of [3H]methylated product. In all cases the substrate dependencies of bursts and methylation rates were proportional to each other. For the perfect 24mer k cat = 0.014/s and K m = 7.7 nM was obtained. In contrast to binary complex formation the two guanine substitutions exerted relatively minor effects on catalytic turnover (the k cat was reduced at most 2. 5-fold), while the two adenine substitutions showed stronger effects (5- to 15-fold reduction in k cat). The effects of base analog substitutions on K m(DNA) were more variable: A-->P (decreased); A-->N and G-->D (unchanged); G-->N (increased).

Phage T4 DNA [N6-adenine] methyltransferase: kinetic studies using oligonucleotides containing native or modified recognition sites.

The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.

Interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with oligonucleotides containing native or modified (defective) recognition sites.

The DNA-[N 6-adenine]-methyltransferase (Dam MTase) of phage T4 catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have used a gel shift assay to monitor complex formation between T4 Dam and various synthetic duplex oligonucleotides, either native or modified/defective. The results are summarized as follows. (i) T4 Dam bound with approximately 100-fold higher affinity to a 20mer specific (GATC-containing) duplex containing the canonical palindromic methylation sequence, GATC, than to a non-specific duplex containing another palindrome, GTAC. (ii) Compared with the unmethylated duplex, the hemimethylated 20mer specific duplex had a slightly increased ( approximately 2-fold) ability to form complexes with T4 Dam. (iii) No stable complex was formed with a synthetic 12mer specific (GATC-containing) duplex, although T4 Dam can methylate it. This indicates that there is no relation between formation of a catalytically competent 12mer-Dam complex and one stable to gel electrophoresis. (iv) Formation of a stable complex did not require that both strands be contiguous or completely complementary. Absence of a single internucleotide phosphate strongly reduced complex formation only when missing between the T and C residues. This suggests that if T4 Dam makes critical contact(s) with a backbone phosphate(s), then the one between T and C is the only likely candidate. Having only one half of the recognition site intact on one strand was sufficient for stable complex formation provided that the 5'G.C base-pairs be present at both ends of the palindromic, GATC. Since absence of either a G or C abolished T4 Dam binding, we conclude that both strands are recognized by T4 Dam.

Comparative studies of the phage T2 and T4 DNA (N6-adenine)methyltransferases: amino acid changes that affect catalytic activity.

The bacteriophage T2 and T4 dam genes code for a DNA (N6-adenine)methyltransferase (MTase). Nonglucosylated, hydroxymethylcytosine-containing T2gt- virion DNA has a higher level of methylation than T4gt- virion DNA does. To investigate the basis for this difference, we compared the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases. Results from Western blotting (immunoblotting) showed that the same amounts of MTase protein were produced after infection with T2 and T4. Kinetic analyses with purified homogeneous enzymes showed that the two MTases had similar Km values for the methyl donor, S-adenosyl-L-methionine, and for substrate DNA. In contrast, they had different k(cat) values (twofold higher for T2 Dam MTase). We suggest that this difference can account for the ability of T2 Dam to methylate viral DNA in vivo to a higher level than does T4 Dam. Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 [T4, Ser; T2, Pro], 26 [T4, Asn; T2, Asp], and 188 [T4, Asp; T2, Glu]), we have produced hybrid proteins to determine which residue(s) is responsible for increased catalytic activity. The results of these analyses showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and noncanonical sites. Moreover, a single substitution of either residue 20 or 26 was sufficient to increase the k(cat) of T4 Dam.

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.

Phage T4 DNA [N6-adenine]methyltransferase. Overexpression, purification, and characterization.

The bacteriophage T4 dam gene, encoding the Dam DNA [N6-adenine]methyltransferase (MTase), has been subcloned into the plasmid expression vector, pJW2. In this construct, designated pINT4dam, transcription is from the regulatable phage lambda pR and pL promoters, arranged in tandem. A two-step purification scheme using DEAE-cellulose and phosphocellulose columns in series, followed by hydroxyapatite chromatography, was developed to purify the enzyme to near homogeneity. The yield of purified protein was 2 mg/g of cell paste. The MTase has an s20,w of 3.0 S and a Stokes radius of 23 A and exists in solution as a monomer. The Km for the methyl donor, S-adenosylmethionine, is 0.1 x 10(-6) M, and the Km for substrate nonglucosylated, unmethylated T4 gt- dam DNA is 1.1 x 10(-12) M. The products of DNA methylation, S-adenosyl-L-homocysteine and methylated DNA, are competitive inhibitors of the reaction; Ki values of 2.4 x 10(-6) M and 4.6 x 10(-12) M, respectively, were observed. T4 Dam methylates the palindromic tetranucleotide, GATC, designated the canonical sequence. However, at high MTase:DNA ratios, T4 Dam can methylate some noncanonical sequences belonging to GAY (where Y represents cytosine or thymine).

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.

Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N6-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding.

Comparison of the deduced amino acid sequences of DNA-[N6-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY [or closely related] motif. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif [located in conserved region IV of the T4 Dam-MTase] to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type [wt] the two mutant enzymic forms had: (i) an increased [5 and 20-fold, respectively] Km for substrate, S-adenosyl-methionine [AdoMet]; (ii) a slightly reduced [2 and 4-fold lower] kcat; (iii) a strongly reduced kcat/KmAdoMet [10 and 100-fold]; and (iv) almost the same Km for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced [4 and 9-fold lower] Ka for AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains or is part of an AdoMet-binding site.

Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding.

Comparison of the deduced amino acid sequences of DNA-[N(6)-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY-motif, or a variant of it. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif (located in conserved region IV of the T4 Dam-MTase) to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type (wt) the two mutant enzymic forms had: (i) an increased (6 and 23-fold, respectively) K(m) for substrate, S-adenosyl-methionine (AdoMet) and an increased (6 and 23-fold) K(i) for product, S-adenosyl-homocysteine (AdoHcy); (ii) a slightly reduced (1.5 and 3-fold lower) k(cat); (iii) a strongly reduced k(cat)/K(m) (AdoMet) (10 and 80-fold); and (iv) the same K(m) for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced (3 and 7-fold lower) K(a) for AdoMet; all forms bound two molecules of AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains an AdoMet-binding site.

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.

Analysis of nuclear sequences homologous to the B4 plasmid-like DNA of rice mitochondria; evidence for sequence transfer from mitochondria to nuclei.

Nuclear sequences homologous to the plasmid-like DNA, B4, were analyzed in the Japonica rice variety, Fujiminori. Homologous sequences existed at several positions in the nuclear genome, but each contained only a portion of the B4 sequence. It was impossible to reconstruct the entire sequence of B4 even by collating all the homologous sequences. Overlaps between some of the B4 sequences present in the nuclear genome resulted in parts of the sequence being represented more than once. These features indicate that nuclear sequences homologous to B4 are not the origin of B4 and that they have been transferred from mitochondria and integrated into the nuclear genome. Five other foreign sequences originating in the chloroplast or mitochondrial genome were found within 1 kb of the B4-homologous sequences. Structural analysis is consistent with the hypothesis that the DNA sequences were transferred via RNA.

Nucleotide sequence of the EcoRII restriction endonuclease gene.

The nucleotide sequence of a 1394 basepair (bp) DNA fragment containing the EcoRII restriction endonuclease (R.EcoRII) gene was determined. The endonuclease gene is 1206 bp in length (predicted 402 amino acids (aa) and Mr = 45 178) and is separated by 33 bp from the EcoRII modification methylase (M.EcoRII) gene. The EcoRII restriction-modification system has a tail-to-tail organization of the two genes.

Trans-dominant mutation affecting restriction and modification in Escherichia coli K-12.

We have analysed the mechanism of action of a ts mutation in E. coli, which has an effect on the expression of the restriction and modification phenotype. The frequencies of recombinants obtained in transduction experiments support the idea that the temperature sensitive mutation is located outside the hsd operon in the gene denoted hsd.X. Complementation experiments demonstrated the trans-dominant nature of the temperature sensitive mutation. The possible role of the hsd.X product in the formation of EcoR.K and EcoM.K complexes and their interaction with the recognition site on the DNA is discussed.