N6-methyladenine Research Articles

Functional organization of box C/D RNA-guided RNA methyltransferase.

Box C/D RNA protein complexes (RNPs) catalyze site-specific 2′-O-methylation of RNA with specificity determined by guide RNAs. In eukaryotic C/D RNP, the paralogous Nop58 and Nop56 proteins specifically associate with terminal C/D and internal C'/D' motifs of guide RNAs, respectively. We have reconstituted active C/D RNPs with recombinant proteins of the thermophilic yeast Chaetomium thermophilum. Nop58 and Nop56 could not distinguish between the two C/D motifs in the reconstituted enzyme, suggesting that the assembly specificity is imposed by trans-acting factors in vivo. The two C/D motifs are functionally independent and halfmer C/D RNAs can also guide site-specific methylation. Extensive pairing between C/D RNA and substrate is inhibitory to modification for both yeast and archaeal C/D RNPs. N6-methylated adenine at box D/D' interferes with the function of the coupled guide. Our data show that all C/D RNPs share the same functional organization and mechanism of action and provide insight into the assembly specificity of eukaryotic C/D RNPs.

Human and Arabidopsis alpha-ketoglutarate-dependent dioxygenase homolog proteins-New players in important regulatory processes.

The family of AlkB homolog (ALKBH) proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate (2OG), and Fe(II)-dependent dioxygenase are involved in a number of important regulatory processes in eukaryotic cells including repair of alkylation lesions in DNA, RNA, and nucleoprotein complexes. There are nine human and thirteen Arabidopsis thaliana ALKBH proteins described, which exhibit diversified functions. Among them, human ALKBH5 and FaT mass and Obesity-associated (FTO) protein and Arabidopsis ALKBH9B and ALKBH10B have been recognized as N6 methyladenine (N6 meA) demethylases, the most abundant posttranscriptional modification in mRNA. The FTO protein is reported to be associated with obesity and type 2 diabetes, and involved in multiple other processes, while ALKBH5 is induced by hypoxia. Arabidopsis ALKBH9B is an N6 meA demethylase influencing plant susceptibility to viral infections via m6 A/A ratio control in viral RNA. ALKBH10B has been discovered to be a functional Arabidopsis homolog of FTO; thus, it is also an RNA N6 meA demethylase involved in plant flowering and several other regulatory processes including control of metabolism. High-throughput mass spectrometry showed multiple sites of human ALKBH phosphorylation. In the case of FTO, the type of modified residue decides about the further processing of the protein. This modification may result in subsequent protein ubiquitination and proteolysis, or in the blocking of these processes. However, the impact of phosphorylation on the other ALKBH function and their downstream pathways remains nearly unexplored in both human and Arabidopsis. Therefore, the investigation of evolutionarily conserved functions of ALKBH proteins and their regulatory impact on important cellular processes is clearly called for.

Selectively Disrupting m6A-Dependent Protein-RNA Interactions with Fragments.

We report a crystallographic analysis of small-molecule ligands of the human YTHDC1 domain that recognizes N6-methylated adenine (m6A) in RNA. The 30 binders are fragments (molecular weight < 300 g mol-1) that represent 10 different chemotypes identified by virtual screening. Despite the structural disorder of the binding site loop (residues 429-439), most of the 30 fragments emulate the two main interactions of the -NHCH3 group of m6A. These interactions are the hydrogen bond to the backbone carbonyl of Ser378 and the van der Waals contacts with the tryptophan cage. Different chemical groups are involved in the conserved binding motifs. Some of the fragments show favorable ligand efficiency for YTHDC1 and selectivity against other m6A reader domains. The structural information is useful for the design of modulators of m6A recognition by YTHDC1.

Mammalian ALKBH1 serves as an N6-mA demethylase of unpairing DNA

N6-methyladenine (N6-mA) of DNA is an emerging epigenetic mark in mammalian genome. Levels of N6-mA undergo drastic fluctuation during early embryogenesis, indicative of active regulation. Here we show that the 2-oxoglutarate-dependent oxygenase ALKBH1 functions as a nuclear eraser of N6-mA in unpairing regions (e.g., SIDD, Stress-Induced DNA Double Helix Destabilization regions) of mammalian genomes. Enzymatic profiling studies revealed that ALKBH1 prefers bubbled or bulged DNAs as substrate, instead of single-stranded (ss-) or double-stranded (ds-) DNAs. Structural studies of ALKBH1 revealed an unexpected “stretch-out” conformation of its “Flip1” motif, a conserved element that usually bends over catalytic center to facilitate substrate base flipping in other DNA demethylases. Thus, lack of a bending “Flip1” explains the observed preference of ALKBH1 for unpairing substrates, in which the flipped N6-mA is primed for catalysis. Co-crystal structural studies of ALKBH1 bound to a 21-mer bulged DNA explained the need of both flanking duplexes and a flipped base for recognition and catalysis. Key elements (e.g., an ALKBH1-specific α1 helix) as well as residues contributing to structural integrity and catalytic activity were validated by structure-based mutagenesis studies. Furthermore, ssDNA-seq and DIP-seq analyses revealed significant co-occurrence of base unpairing regions with N6-mA in mouse genome. Collectively, our biochemical, structural and genomic studies suggest that ALKBH1 is an important DNA demethylase that regulates genome N6-mA turnover of unpairing regions associated with dynamic chromosome regulation.

N6-methylated adenine on the target sites of mamA from Mycobacterium bovis BCG enhances macrophage activation by CpG DNA in mice

CpG motifs in DNA sequences are recognized by Toll-like receptor 9 and activate immune cells. Bacterial genomic DNA (gDNA) has modified cytosine bases (5-methylcytosine [5 mC]) and modified adenine bases (6-methyladenine [6 mA]). 5 mC inhibits immune activation by CpG DNA; however, it is unclear whether 6 mA inhibits immune activation by CpG DNA. Mycobacterium bovis BCG (BCG) has three adenine methyltransferases (MTases) that act on specific target sequences. In this study, we examined whether the 6 mA at the target sites of adenine MTases affected the immunostimulatory activity of CpG DNA. Our results showed that only 6 mA located at the target sequence of mamA, an adenine MTase from BCG, enhanced interleukin (IL)-12p40 production from murine bone marrow-derived macrophages (BMDMs) stimulated with CpG DNA. Enhancement of IL-12p40 production in BMDMs was also observed when BMDMs were stimulated with CpG DNA ligated to oligodeoxynucleotides (ODNs) harboring 6 mA. Accordingly, we then evaluated whether gDNA from adenine MTase-deficient BCG was less efficient with regard to stimulation of BMDMs. Indeed, gDNA from a mamA-deficient BCG had less ability to activate BMDMs than that from wild-type BCG. We concluded from these results that adenine methylation on ODNs and bacterial gDNA may enhance immune activity induced by CpG DNA.

M6A RNA Degradation Products Are Catabolized by an Evolutionarily Conserved N6-Methyl-AMP Deaminase in Plant and Mammalian Cells

N6-methylated adenine (m6A) is the most frequent posttranscriptional modification in eukaryotic mRNA. Turnover of RNA generates N6-methylated AMP (N6-mAMP), which has an unclear metabolic fate. We show that Arabidopsis thaliana and human cells require an N6-mAMP deaminase (ADAL, renamed MAPDA) to catabolize N6-mAMP to inosine monophosphate in vivo by hydrolytically removing the aminomethyl group. A phylogenetic, structural, and biochemical analysis revealed that many fungi partially or fully lack MAPDA, which coincides with a minor role of N6A-RNA methylation in these organisms. MAPDA likely protects RNA from m6A misincorporation. This is required because eukaryotic RNA polymerase can use N6-mATP as a substrate. Upon abrogation of MAPDA, root growth is slightly reduced, and the N6-methyladenosine, N6-mAMP, and N6-mATP concentrations are increased in Arabidopsis. Although this will potentially lead to m6A misincorporation into RNA, we show that the frequency is too low to be reliably detected in vivo. Since N6-mAMP was severalfold more abundant than N6-mATP in MAPDA mutants, we speculate that additional molecular filters suppress the generation of N6-mATP. Enzyme kinetic data indicate that adenylate kinases represent such filters being highly selective for AMP versus N6-mAMP phosphorylation. We conclude that a multilayer molecular protection system is in place preventing N6-mAMP accumulation and salvage.

The Presence of N6‐methyladenine and 5‐methylcytosine in Trypanosoma brucei RNA

Trypanosoma brucei is the parasite responsible for Human African Trypanosomiasis, commonly referred to as African Sleeping Sickness. In T. brucei there is a lack of promoters and other regulatory sequences normally present in DNA. Thus, posttranscriptional RNA alterations such as methylation may be important in this organism. To investigate the occurrence of RNA base methylation, a series of ELISAs for N6‐methyladenine (N6‐MeA) and 5‐methylcytosine (5‐MeC) were performed. The presence of N6‐MeA in total RNA and small RNA was detected with minimal differences between procyclic and bloodstream forms. ELISAs identified 5‐MeC in both procyclic and bloodstream form total RNA samples, and low 5‐MeC levels in small RNA. Using a robust bisulfite sequencing technique, total RNA and small RNA 5‐MeC residues were mapped with base resolution. There was an elevated frequency of 5‐MeC in tRNA with numerous residues having a level of methylation greater than 50%. To investigate the source of 5‐MeC in RNA, BLAST identified seven putative T. brucei cytosine RNA methyltransferases (TbCRMTs). Of the seven TbCRMT RNAi lines generated, 2 of the 7 resulted in growth defects when the RNAi system was activated. To study the TbCRMTs in vitro, recombinant pET28‐TbCRMT plasmids were introduced into E. coli. The TbCRMTs were expressed as N‐terminal 6x‐his tagged proteins and characterized by SDS‐PAGE and western blot analysis. The TbCRMTs are soluble and, in the future, successful protein purification will enable in vitro RNA methylation assays. In summary, the data indicate that T. brucei transcriptome contains modified RNA bases, and we have identified the 5‐MeC positions and putative enzymes responsible.Support or Funding InformationNIH 1R15AI13342801This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vibrational fingerprints of N6-methyladenine and N6,N6-dimethyladenine in Raman spectra

Vibrational fingerprints of N6-methyladenine (NMA) and N6,N6-dimethyladenine (NDMA) on gold nanoparticles (AuNPs) were investigated by means of Raman spectroscopy. NMA and NDMA have one methyl group and two methyl groups, respectively, at the amino moiety of the adenine purine unit. Surface-enhanced Raman scattering (SERS) spectra were analyzed to explain the difference in the spectral features among adenine, NMA, and NDMA based on density functional theory (DFT) calculations on gold clusters. NMA has unique bands at 717 and 1101cm−1 in their SERS spectra on AuNPs, which can be differentiated from those of adenine. After observing these unique spectra features, the SERS spectra of NMA and NDMA on AuNPs were dissimilar, suggesting that the methyl group at the amino groups of the adenine may be significantly affecting the binding on AuNPs, as supported by DFT calculations of NMA and NDMA on gold clusters. Our Raman study indicates that the methylation at the amino group in adenine may play a significant role in providing vibrational finger prints in the SERS spectra on metal surfaces.

DNA N6-methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs.

ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine (N6-mA), a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells (MSCs) was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBH1-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation.

Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets

Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (∼2-fold; p ≤ 0.05). Using 3-MA as a ‘model’ monomethyladenine, it was found that 3-MA augmented [cAMP]i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3′K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]i and then potentiate glucose-induced insulin secretion and production in parallel.

Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations.

Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

Direct surface-enhanced Raman scattering analysis of DNA duplexes.

The exploration of the genetic information carried by DNA has become a major scientific challenge. Routine DNA analysis, such as PCR, still suffers from important intrinsic limitations. Surface-enhanced Raman spectroscopy (SERS) has emerged as an outstanding opportunity for the development of DNA analysis, but its application to duplexes (dsDNA) has been largely hampered by reproducibility and/or sensitivity issues. A simple strategy is presented to perform ultrasensitive direct label-free analysis of unmodified dsDNA with the means of SERS by using positively charged silver colloids. Electrostatic adhesion of DNA promotes nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at nanogram level. As potential applications, we report the quantitative recognition of hybridization events as well as the first examples of SERS recognition of single base mismatches and base methylations (5-methylated cytosine and N6-methylated Adenine) in duplexes.

Discrimination of N6-methyl adenine in a specific DNA sequence

We have developed a novel chemistry-based method to detect adenine methylation. A DNA probe functionalized with a formyl group discriminates between adenine and N6-methyl adenine by the formation of a selective interstrand cross-link.

Undetectable levels of N6-methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase

Three methylated bases, 5-methylcytosine, N4-methylcytosine and N6-methyladenine (m6A), can be found in DNA. However, to date, only 5-methylcytosine has been detected in mammalian genomes. To reinvestigate the presence of m6A in mammalian DNA, we used a highly sensitive method capable of detecting one N6-methyldeoxyadenosine per million nucleosides. Our results suggest that the total mouse genome contains, if any, less than 10 3 m6A. Experiments were next performed on PRED28, a putative mammalian N6-DNA methyltransferase. The murine PRED28 encodes two alternatively spliced RNA. However, although recombinant PRED28 proteins are found in the nucleus, no evidence for an adenine-methyltransferase activity was detected.

Effect of Methylated Adenine in Plasmid DNA on Transgene Expression in Mice

Cationic lipid-mediated transfer of DNA is promising in gene therapy. However, one disadvantage with this approach is the induction of an inflammatory response, which may decrease transgene expression. Recently, we found that plasmid DNA containing N6-methyladenine (N6-MeA), a bacterium-specific modified base, induced cytokine twice as efficiently as plasmid DNA without N6-MeA, when complexed with cationic lipids. Thus, plasmid DNA without N6-MeA might express a transgene more efficiently than that containing N6-MeA in vivo. To evaluate the effects of adenine methylation on transgene expression in vivo, we injected luciferase-encoding plasmid DNA, complexed with cationic lipids or a cationic polymer, intravenously into mice. When the plasmid DNA-cationic lipid complexes were injected, the luciferase expression from the methylated and unmethylated plasmids was similar, although cytokine was more efficiently elicited by the methylated DNA than the unmethylated DNA. Hydrodynamics-based injections of plasmid DNA-cationic polymer complexes did not induce cytokine, and the luciferase expression from the unmethylated plasmid was slightly lower than that from the methylated plasmid DNA. These results suggest that the presence of N6-MeA did not reduce transgene expression in vivo.

Structure and Dynamics of Hemimethylated GATC Sites: IMPLICATIONS FOR DNA-SeqA RECOGNITION

DNA methylation occurs in most organisms from bacteria to mammals and provides a mechanism for epigenetic control of a variety of cellular processes. In Escherichia coli, most of the N6 positions in adenines found in the sequence GATC are methylated by DNA adenine methyltransferase. After DNA replication, the GATC sites exist transiently in the hemimethylated state, and the specific recognition of these hemimethylated GATC sites is essential for several processes, including sequestration of the site of replication initiation by the SeqA protein, strand discrimination in DNA mismatch repair by the MutH protein, and transcription of several genes. Here, we characterize the solution structure and dynamics of two dodecamer DNA duplexes that each contains a single GATC site in either unmethylated or hemimethylated state. We found that the N6-methylated adenine of a hemimethylated GATC site undergoes a slow trans-cis interconversion. The release of a tightly bound cation from hemimethylated DNA explains the instability of this structure. In addition, quantitative structural analysis revealed that hemimethylated DNA has unusual backbone structures and a remarkably narrow major groove. These dynamic and structural features provide insights into the specific recognition of hemimethylated GATC sites by the SeqA protein.

The HaeIV restriction modification system of Haemophilus aegyptius is encoded by a single polypeptide

The HaeIV restriction endonuclease (ENase) belongs to a distinct class of ENases, characterized by its ability to cleave double-stranded DNA on both sides of its recognition sequence, excising a short DNA fragment that includes the recognition sequence. The gene encoding the HaeIV ENase was cloned from Haemophilus aegyptius into pUC19 using a previously described system that does not need the knowledge that a particular ENase is produced by a bacterial strain. DNA sequence analysis of the insert contained on this plasmid identified a single open reading frame (ORF), with the predicted protein having an apparent molecular mass of ∼110 kDa. The protein encoded by this ORF was purified to homogeneity from Escherichia coli strain ER1944 carrying the haeIVRM gene on a recombinant plasmid under the control of the inducible ara promoter. The protein possessed both ENase and methyltransferase (MTase) activities. Amino acid sequence analysis was able to identify several conserved motifs found in DNA MTases, located in the middle of the protein. The enzyme recognizes the interrupted palindromic sequence 5′ GAPyNNNNNPuTC 3′, cleaving double-stranded DNA on both strands upstream and downstream of the recognition sequence, releasing an ∼33 bp fragment. The ENase possessed an absolute requirement only for Mg+2. ATP had no influence on ENase or MTase activities. The ENase made the first strand cleavage randomly on either side of the recognition sequence, but the second cleavage occurred more slowly. The MTase activity modified symmetrically located adenine residues on both strands within the recognition sequence yielding N6-methyl adenine. Furthermore, the MTase was active as a dimer.

"Small is beautiful": major modifications in DNA structure or dynamics by small substituents or ligands.

This short review assembles the contributions of the author's laboratory to the structural aspects of DNA. DNA was modified by small ligands and/or substituents. There are three aspects to this work: a) Protonation of guanosine and DNA and the formation of triple- and quadruple-strands of guanosine, its nucleotides, their polymers and DNA. b) Substitution of the 2'-position of deoxyribose by the most polar atom, fluorine: studies on 2'-deoxy-2'-fluro-nucleosides, -nucleotides and their polymers, studied both by structural and biological methods. c) The effect of introducing the methyl group in the large groove of DNA: NMR studies of oligonucleotides containing N6-methylated adenine residues, and enzymatic and molecular biology work on Dam methylase are reported.

DNA methylation in leprosy-associated bacteria: Mycobacterium leprae and Corynebacterium tuberculostearicum

The DNAs of two kinds of microorganisms from human leprosy lesion, Mycobacterium leprae and Corynebacterium tuberculostearicum (also known as "leprosy-derived corynebacterium" or LDC), have been analysed and compared with the genomes of reference bacteria of the CMN group (genera Corynebacterium, Mycobacterium and Nocardia). The guanine-plus-cytosine content (% GC) of DNA was determined by a double-labelling procedure, which is unaffected by the presence of modified and unusual bases (that alter both buoyant density and mid-melting-point determinations). Accordingly, the DNAs of seven LDC strains had GC values of 54-56 mol %, and that of armadillo-grown M. leprae a value of 54.8 +/- 0.9 mol %. Restriction patterns disclosed no methylated cytosine in the DNA sequences CCGG, GGCC, AGCT and GATC of either LDC or M. leprae DNA. N6-methyl adenine was present in the sequence GATC of all LDC strains, but was missing from the genomes of all others CMN organisms analysed, including M. leprae. By HPLC analysis of LDC-DNA hydrolysates, it was found that N6-methyladenine amounted to 1.8% of total DNA adenine, and was present exclusively within GATC sequences, which appeared all to be methylated. It is concluded that LDC represent a group of corynebacteria endowed with high genetic homogeneity and a unique restriction pattern, whereby their genome is easily distinguished from that of M. leprae, which has a similar base composition.

Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12.

Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N(6)-methyladenine (N(6)-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict lambda. None of the mutations had the effect of being lethal.