Samuel L. Schlagman

Structure of the bacteriophage T4 DNA adenine methyltransferase

DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a β-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.

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.

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.

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 K m for substrate (AdoMet), but a decrease in the K m for substrate DNA. This is reflected in minor changes in k cat/K m for DNA, but in 10‐ to 100‐fold reductions in k cat/K m 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.

The UV excision-repair system of Saccharomyces cerevisiae is involved in the removal of methylcytosines formed in vivo by a cloned prokaryotic DNA methyltransferase

SummaryDNA methyltransferase activity is not normally found in yeast. To investigate the response of Saccharomyces cerevisiae to the presence of methylated bases, we introduced the Bacillus subtilis SPR phage DNA-[cytosine-5] methyltransferase gene on the shuttle vector, YEp51. The methyltransferase gene was functionally expressed in yeast under the control of the inducible yeast GAL10 promoter. Following induction we observed a time-dependent methylation of yeast DNA in RAD+ and rad2 mutant strains; the rad2 mutant is defective in excision-repair of UV-induced DNA damage. Analysis of restriction endonuclease digestion patterns revealed that the relative amount of methylated DNA was greater in the excision defective rad2 mutant than in the RAD+ strain. These data indicate that the yeast excision-repair system is capable of recognizing and removing m5C residues.

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.

Effect of N6-methyladenine (m6A) content in DNA on spontaneous reversion in non-glucosylated phage T2 gt—: Evidence for base analog mutagen activity

Bacteriophage T2 is unusual in that its DNA contains 5-hydroxymethyl-cytosine (hm5C) in place of cytosine; in addition, hm5C is further modified by glucosylation (Revel and Luria, 1970). Phage T2 DNA also contains the methylated base, N6-methyladenine (m6A), and these are produced as a post-replicational modification by a phage induced DNA adenine methylase. Mutants which do not glucosylate their DNA, designated gt−, have been isolated. From these it has been possible to obtain mutants in the gene (dam) specifying the phage DNA methylase activity. Thus, parental phage T2 gt− dam+ and successive mutant strains, T2gt−damh and T2gt−damh dam-1, differ in their m6A contents; respectively 0.7, 2.3 and 0.05% of the adenine residues are m6A (Hattman, 1970; Revel and Hattman, 1971). Apparently the host enzyme is unable to methylate hm5C-containing DNA. Whereas the host DNA adenine methylase recognizes the sequence, G-A-T-C (Lacks and Greenberg, 1977; Hattman et al., 1978a), the phage dam+ and damh enzymes appear capable of methylating G-A-T and G-A-(T/C), respectively (Hattman et al., 1978b; Brooks and Hattman, 1978).