T4 Dam is a DNA adenine methyltransferase (EC 2.1.1.72) that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of adenine within GATC palindromic sequences. The enzyme functions as a monomer and methylates both cytosine- and 5-hydroxymethylcytosine-containing DNA substrates. The primary biological role is to protect phage T4 genomic DNA from degradation by the host E. coli restriction-modification defense system. This is a well-characterized "orphan" methyltransferase with high-resolution crystal structures available (PDB: 1Q0S, 1Q0T, 1YF3).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
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GO:0003676
nucleic acid binding
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IEA
GO_REF:0000002 |
MODIFY |
Summary: T4 Dam binds DNA as part of its methyltransferase activity. Crystal structures confirm DNA binding capability [PMID:12937411]. However, "nucleic acid binding" is overly general for a protein with well-characterized DNA-specific binding and enzymatic activity.
Reason: This term is too general. The protein specifically binds DNA (not RNA) in a sequence-specific manner (GATC recognition). More specific terms like GO:0003677 (DNA binding) or GO:0043565 (sequence-specific DNA binding) are more informative and are already annotated.
Proposed replacements:
sequence-specific DNA binding
Supporting Evidence:
PMID:12937411
T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain
file:BPT4/DAM/DAM-deep-research-falcon.md
Crystal structures resolved a binary complex (Dam-AdoHcy) and a ternary Dam-DNA-AdoHcy complex
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GO:0003677
DNA binding
|
IEA
GO_REF:0000043 |
KEEP AS NON CORE |
Summary: T4 Dam binds double-stranded DNA containing GATC sequences. This is well established by crystal structures showing Dam-DNA complexes [PMID:12937411, PMID:15882618] and biochemical studies demonstrating Km for DNA substrate of 1.1 x 10^-12 M [PMID:7782299]. The term is accurate, but it is a broad parent of the more informative DNA methyltransferase functions.
Reason: DNA binding is necessary for Dam activity, but it is not the best core annotation for this enzyme. The core function is site-specific DNA adenine methyltransferase activity, with DNA binding as a supporting molecular property.
Supporting Evidence:
PMID:7782299
the Km for substrate nonglucosylated, unmethylated T4 gt- dam DNA is 1.1 x 10(-12) M
PMID:12937411
a DNA binding domain consisting of a five-helix bundle and a beta-hairpin
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GO:0006260
DNA replication
|
IEA
GO_REF:0000043 |
REMOVE |
Summary: While T4 Dam methylates newly replicated phage DNA, the enzyme itself is not directly involved in DNA replication (DNA synthesis). Dam acts on DNA after or during replication to provide methylation-based protection, but does not participate in the replication machinery itself.
Reason: T4 Dam is not part of the DNA replication machinery. It is a DNA modification enzyme that acts on replicated DNA. The methylation protects DNA from restriction enzymes but does not contribute to DNA synthesis. This appears to be an over-annotation based on the UniProtKB keyword "DNA replication" which may have been applied due to the gene's role in the phage replication cycle rather than the enzymatic mechanism of replication.
Supporting Evidence:
PMID:2510127
Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam)
file:BPT4/DAM/DAM-deep-research-falcon.md
T4 Dam is a DNA N6-adenine methyltransferase that transfers a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of adenine within GATC sites
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GO:0008168
methyltransferase activity
|
IEA
GO_REF:0000120 |
KEEP AS NON CORE |
Summary: T4 Dam is indeed a methyltransferase, specifically a DNA adenine methyltransferase (EC 2.1.1.72). This is experimentally validated [PMID:2510127, PMID:7782299].
Reason: This is accurate but quite general. The more specific term GO:0009007 (site-specific DNA-methyltransferase adenine-specific activity) is also annotated and is more precise. Keeping this general term as a non-core parent is acceptable because it is not incorrect, but it should not be treated as the main curated function.
Supporting Evidence:
PMID:7782299
The bacteriophage T4 dam gene, encoding the Dam DNA [N6-adenine]methyltransferase (MTase)
PMID:2510127
Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam)
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GO:0009007
site-specific DNA-methyltransferase (adenine-specific) activity
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: T4 Dam specifically methylates the N6 position of adenine within GATC sequences. This site-specific adenine methylation activity is the core molecular function of the enzyme, experimentally demonstrated in multiple studies [PMID:2510127, PMID:7782299].
Reason: This is the most precise and accurate molecular function term for T4 Dam. The enzyme recognizes the GATC palindrome and methylates the adenine at N6 position. This is the core enzymatic function and should be retained.
Supporting Evidence:
PMID:7782299
T4 Dam methylates the palindromic tetranucleotide, GATC, designated the canonical sequence
PMID:2510127
Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam) which recognizes primarily the sequence GATC
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GO:0009307
DNA restriction-modification system
|
IEA
GO_REF:0000002 |
MODIFY |
Summary: T4 Dam is specifically an "orphan" methyltransferase - it is part of a modification system but lacks a cognate restriction enzyme. The phage uses Dam to protect its DNA FROM host restriction systems, not as part of a complete R-M system of its own.
Reason: This term implies Dam is part of a complete restriction-modification system with both restriction and modification activities. T4 Dam is explicitly an orphan methyltransferase (M.EcoT4Dam) that provides DNA protection without an associated restriction enzyme. The appropriate term is GO:0099018 (symbiont-mediated evasion of host restriction-modification system) which accurately describes the protective role against host R-M systems.
Proposed replacements:
symbiont-mediated evasion of host restriction-modification system
Supporting Evidence:
file:BPT4/DAM/DAM-deep-research-falcon.md
T4 Dam is explicitly an orphan methyltransferase (M.EcoT4Dam) that provides DNA protection without an associated restriction enzyme
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GO:0016740
transferase activity
|
IEA
GO_REF:0000043 |
KEEP AS NON CORE |
Summary: T4 Dam catalyzes transfer of a methyl group from AdoMet to DNA adenine, which is a transferase reaction. This is accurate but extremely general.
Reason: While accurate, this is a very high-level term. More specific terms (GO:0008168 methyltransferase activity, GO:0009007 site-specific DNA-methyltransferase activity) are also present and more informative. Transferase activity is not incorrect, but it should be retained only as a non-core parent term.
Supporting Evidence:
PMID:7782299
The bacteriophage T4 dam gene, encoding the Dam DNA [N6-adenine]methyltransferase (MTase)
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GO:0032259
methylation
|
IEA
GO_REF:0000120 |
MODIFY |
Summary: T4 Dam catalyzes DNA adenine methylation. The enzyme is involved in the biological process of DNA methylation to protect phage DNA.
Reason: The annotation is directionally correct but too broad. T4 Dam modifies DNA by site-specific adenine methylation; the best available non-obsolete process replacement is GO:0006304 DNA modification, while the phage-specific outcome is captured by GO:0099018 restriction-modification system evasion.
Proposed replacements:
DNA modification
Supporting Evidence:
PMID:2510127
Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam)
|
|
GO:0052031
symbiont-mediated perturbation of host defense response
|
IEA
GO_REF:0000043 |
MODIFY |
Summary: This term and its definition ("A process in which a symbiont interferes with the ability of the host to mount a defense in response to its presence") is too broad for the specific biology. T4 Dam does not generally perturb host defense; it methylates phage DNA so the genome can evade host restriction- modification systems.
Reason: The broad host-defense framing should be replaced by the mechanistic phage term. The correct term for T4 Dam's role in protecting phage DNA from bacterial restriction enzymes is GO:0099018 (symbiont-mediated evasion of host restriction-modification system), which is already annotated and explicitly covers phages and bacterial restriction systems.
Proposed replacements:
symbiont-mediated evasion of host restriction-modification system
Supporting Evidence:
file:BPT4/DAM/DAM-deep-research-falcon.md
By methylating GATC, T4 Dam participates in phage counter-defense against host restriction systems
|
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GO:0052170
symbiont-mediated suppression of host innate immune response
|
IEA
GO_REF:0000043 |
MODIFY |
Summary: This term is defined as "A process in which a symbiont inhibits or disrupts the normal execution of the innate immune response of the host organism, the host's first line of defense against infection." This is the wrong level of abstraction for T4 Dam. The supported mechanism is restriction-modification evasion by phage DNA methylation, not general suppression of an innate immune response.
Reason: This SPKW-derived annotation conflates phage antirestriction with broad immune-response suppression. Bacteriophage T4 infects Escherichia coli, and the evidence points to a specific bacterial restriction-modification counter- defense mechanism. Replace this row with GO:0099018, which is already present and accurately captures the Dam-mediated phage mechanism.
Proposed replacements:
symbiont-mediated evasion of host restriction-modification system
Supporting Evidence:
file:BPT4/DAM/DAM-deep-research-falcon.md
T4 Dam acts in the infected E. coli cytoplasm on phage DNA, methylating GATC sites as the genome is replicated
|
|
GO:0099018
symbiont-mediated evasion of host restriction-modification system
|
IEA
GO_REF:0000043 |
ACCEPT |
Summary: This is the correct term for T4 Dam's biological role. The GO definition explicitly states: "A process by which a symbiont evades the DNA restriction modification system of its host. This process occurs in phages to protect themselves from bacterial restriction enzyme systems. Some viruses encode their own methyltransferase in order to protect their genome from host restriction enzymes." This precisely describes T4 Dam.
Reason: This is the most accurate biological process term for T4 Dam. The enzyme methylates phage DNA to protect it from E. coli restriction enzymes. The term definition explicitly references phages, bacterial restriction systems, and virus-encoded methyltransferases. T4 Dam is a canonical example of this mechanism.
Supporting Evidence:
file:BPT4/DAM/DAM-deep-research-falcon.md
By methylating GATC, T4 Dam participates in phage counter-defense against host restriction systems
PMID:12937411
DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression
|
|
GO:0032259
methylation
|
IMP
PMID:2510127 Single amino acid changes that alter the DNA sequence specif... |
MODIFY |
Summary: This IMP (Inferred from Mutant Phenotype) annotation is based on the Miner et al. 1989 study that characterized dam mutants. The damh mutation (P126S) produces a hypermethylating phenotype, demonstrating the enzyme's role in DNA methylation in vivo.
Reason: This experimental annotation is well supported, but the term itself is broader than the biology tested. The mutant phenotype supports DNA modification by T4 Dam, not methylation in an unrestricted substrate/process sense. Use the more specific non-obsolete process term GO:0006304 DNA modification.
Proposed replacements:
DNA modification
Supporting Evidence:
PMID:2510127
Hypermethylating mutants, damh, exhibit a relaxation in sequence specificity, that is, they are readily able to methylate non-canonical sites
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GO:0009008
DNA-methyltransferase activity
|
IDA
PMID:7782299 Phage T4 DNA [N6-adenine]methyltransferase. Overexpression, ... |
ACCEPT |
Summary: This IDA (Inferred from Direct Assay) annotation is based on the Kossykh et al. 1995 study that overexpressed, purified, and biochemically characterized T4 Dam. The enzyme was shown to have DNA methyltransferase activity with measured kinetic parameters.
Reason: This is high-quality experimental evidence. The study purified T4 Dam to near homogeneity and characterized its methyltransferase activity with precise kinetic measurements (Km for AdoMet = 0.1 uM, Km for DNA substrate = 1.1 x 10^-12 M). This directly demonstrates the core molecular function of T4 Dam.
Supporting Evidence:
PMID:7782299
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
|
Q: What is the precise timing of Dam methylation during phage infection - does it occur co-replicationally or post-replicationally?
Q: Are there E. coli restriction systems that T4 specifically evades through Dam methylation, and which host strains show differential susceptibility to dam- phage?
Experiment: Systematic analysis of T4 dam- phage viability across E. coli strains with different restriction-modification systems to catalog the protective scope of Dam methylation.
Hypothesis: T4 Dam methylation provides selective protection against specific host restriction systems, and dam- phage will show differential viability depending on the host R-M system present.
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
Research plan and verification
We verified the target as DAM from Enterobacteria phage T4 (UniProt P04392), encoding the DNA (N6-adenine) methyltransferase commonly called T4 Dam. Structural and biochemical studies define it as an AdoMet-dependent m6A MTase with canonical motifs (e.g., motif I Phe-X-Gly; motif IV DPPY) in the N6-adenine/SAM-dependent MTase family, matching the domain annotations provided (SAM-dependent_MTases fold; N6-adenine MTase motifs) (Oct 2003, https://doi.org/10.1038/nsb973) (yang2003structureofthe pages 1-3, yang2003structureofthe pages 9-11). All subsequent research and citations refer to this phage T4 Dam enzyme and not to E. coli Dam.
1) Key concepts and definitions with current understanding
- Primary function and reaction: T4 Dam is a DNA N6-adenine methyltransferase that transfers a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of adenine within GATC sites, generating S-adenosyl-L-homocysteine (AdoHcy) (Oct 2003, https://doi.org/10.1038/nsb973; Sep 2004, https://doi.org/10.1023/b:mbil.0000043943.07792.80) (yang2003structureofthe pages 1-3, zinoviev2004molecularenzymologyof pages 1-2).
- Substrate specificity: Wild-type T4 Dam recognizes GATC and methylates the target adenine; variants can relax or alter specificity (see below) (Oct 2003, https://doi.org/10.1038/nsb973; Oct 1989, https://doi.org/10.1093/nar/17.20.8149) (yang2003structureofthe pages 1-3, miner1989singleaminoacid pages 1-3).
- Cofactor usage and mechanism: AdoMet binds in a conserved pocket; structural work captured the product AdoHcy in a closed cofactor pocket. Release/exchange of AdoHcy appears rate-limiting, and the enzyme likely diffuses along DNA between catalytic cycles (“processive” methylation on DNA bearing multiple sites) (Oct 2003, https://doi.org/10.1038/nsb973) (yang2003structureofthe pages 6-8, yang2003structureofthe pages 1-3).
- Conserved motifs/domains: T4 Dam belongs to the alpha group of AdoMet-dependent MTases. Conserved motifs include motif I (Phe32–Ser33–Gly34), motif II (Asp50), motif IV (Asp-Pro-Pro-Tyr), and motif VI (Asp171-Pro172-Pro173). A cofactor-covering element (residues ~175–203, including helix αD1) closes the pocket in the AdoHcy-bound state (Oct 2003, https://doi.org/10.1038/nsb973) (yang2003structureofthe pages 3-4, yang2003structureofthe pages 11-15, yang2003structureofthe pages 1-3).
- Structure: Crystal structures resolved a binary complex (Dam–AdoHcy) and a ternary Dam–DNA–AdoHcy complex. The protein is a monomer comprising a catalytic domain that binds cofactor and a target-recognition domain (TRD) with a 5-helix bundle and a distinctive β-hairpin. In the DNA-bound structure, the complex exhibits nonspecific DNA contacts with two Dam monomers per duplex; specific base-targeting is inferred from catalytic motifs and prior Dam family studies (Oct 2003, https://doi.org/10.1038/nsb973) (yang2003structureofthe pages 1-3, yang2003structureofthe pages 6-8, yang2003structureofthe pages 11-15).
- Biophysical properties: The enzyme mass is ~30.4–30.7 kDa by sequence/ultracentrifugation; it is largely monomeric in solution, but forms higher-order complexes with DNA. With site-containing oligos, a 2:1 enzyme:duplex complex (E2S) was observed (Sep 2004, https://doi.org/10.1023/b:mbil.0000043943.07792.80) (zinoviev2004molecularenzymologyof pages 1-2).
2) Recent developments and latest research (prioritizing 2023–2024)
- Methylation-based engineering strategies: 2024 reviews highlight using DNA methyltransferases to pre-pattern incoming DNA with host-like methylation to evade R–M barriers, enabling transformation and gene transfer across difficult bacteria; MTases are also being repurposed as reporters and in memory circuits in microbial engineering (Jul 2024, https://doi.org/10.1186/s13068-024-02529-x) (won2024emergingmethylationbasedapproaches pages 3-5).
- Phage adenine MTases in defense evasion: A 2024 study on Shewanella phage Thanatos-1 characterized a phage adenine MTase (TH1_126) with a recognized 5′-ATC motif in E. coli, combining nanopore-based 6mA calling and LC-MS/MS quantification, illustrating modern workflows to profile phage-encoded Dam-like enzymes and link methylation to endonuclease/Cas resistance phenotypes (Mar 2024, https://doi.org/10.1101/2024.02.27.582347) (brandt2024discoveryofa pages 4-6).
- Broader context: Reviews from 2023 synthesize Dam’s roles in bacterial epigenetics, replication control, mismatch repair, and virulence regulation, providing updated reference frameworks for Dam-family enzymes and their emerging biomedical potential (Mar 2023, https://doi.org/10.3389/fmicb.2023.1129437) (gao2023bacterialdnamethyltransferase pages 3-4, gao2023bacterialdnamethyltransferase pages 13-14).
3) Current applications and real-world implementations
- Overcoming restriction barriers and enabling genetic access: Implementations leverage MTases (including Dam-family enzymes) to pre-methylate plasmid or genomic DNA to match target organisms, improving DNA transfer and stability. Strategies include co-expressing multiple target MTases in methylase-free E. coli to prepare shuttle DNAs and using MTase fusions as transcription reporters or epigenetic memory components (Jul 2024, https://doi.org/10.1186/s13068-024-02529-x) (won2024emergingmethylationbasedapproaches pages 3-5).
- Phage defense/counter-defense studies: High-throughput sequencing plus LC-MS/MS pipelines quantify 6mA patterns introduced by phage MTases and correlate with resistance to restriction endonucleases and CRISPR-Cas, informing phage genome engineering and anti-defense repertoire characterization (Mar 2024, https://doi.org/10.1101/2024.02.27.582347) (brandt2024discoveryofa pages 4-6).
- Conceptual and translational opportunities: 2023 analyses point to Dam inhibitors as potential antibiotic adjuvants and Dam perturbation as a route to attenuated live vaccines in some bacterial pathogens, emphasizing Dam-family enzymes’ translational promise (Mar 2023, https://doi.org/10.3389/fmicb.2023.1129437) (gao2023bacterialdnamethyltransferase pages 3-4, gao2023bacterialdnamethyltransferase pages 13-14).
4) Expert opinions and analysis from authoritative sources
- Structural mechanism and catalytic cycle: Expert structural work argues that cofactor/product handling is a key kinetic bottleneck (AdoHcy release), with a closed “cofactor cover” element gating exchange; Dam moves processively along DNA to methylate multiple sites, consistent with its role in comprehensive GATC methylation on phage DNA (Oct 2003, https://doi.org/10.1038/nsb973) (yang2003structureofthe pages 6-8, yang2003structureofthe pages 1-3).
- Sequence specificity determinants: Classic and structural studies identify residues in the TRD and adjacent loops as critical for sequence recognition; single amino-acid changes at positions 126–127 can reprogram specificity or hypermethylation, with tangible effects on host restriction sensitivity—supporting a model where Dam specificity tuning is a phage strategy against diverse host restriction systems (Oct 1989, https://doi.org/10.1093/nar/17.20.8149; Oct 2003, https://doi.org/10.1038/nsb973) (miner1989singleaminoacid pages 1-3, yang2003structureofthe pages 3-4).
- Oligomeric state and DNA engagement: Solution biophysics and crystallography agree Dam is monomeric but forms higher-order complexes with DNA; ternary structures with two Dam monomers bound per duplex suggest facilitated diffusion or sliding, aligning with observed processivity (Sep 2004, https://doi.org/10.1023/b:mbil.0000043943.07792.80; Oct 2003, https://doi.org/10.1038/nsb973) (zinoviev2004molecularenzymologyof pages 1-2, yang2003structureofthe pages 6-8).
5) Relevant statistics and data from recent and foundational studies
- Kinetics and cofactor affinity: A measured Km for AdoMet of ~1.4 μM for T4 Dam; substitutions at Pro172 elevated Km for AdoMet by 5–20× and reduced activity unless AdoMet was increased, indicating motif VI’s role in cofactor binding and catalysis (Oct 1989, https://doi.org/10.1093/nar/17.20.8149; Oct 2003, https://doi.org/10.1038/nsb973) (miner1989singleaminoacid pages 3-5, yang2003structureofthe pages 3-4).
- Processivity and rate limitation: Structural/biochemical analysis supports that AdoHcy release is rate-limiting and that Dam can diffuse along DNA to methylate multiple sites, consistent with processive behavior on multi-GATC substrates (Oct 2003, https://doi.org/10.1038/nsb973) (yang2003structureofthe pages 6-8).
- Mass and oligomerization: Native molecular mass ~30.69 kDa by ultracentrifugation (vs 30.4 kDa by sequence); Dam–DNA complexes can assemble as E2S (two enzymes:one duplex) under certain conditions (Sep 2004, https://doi.org/10.1023/b:mbil.0000043943.07792.80) (zinoviev2004molecularenzymologyof pages 1-2).
- Specificity-altering mutations and anti-restriction: A Pro126→Ser mutation (damh) relaxes specificity, enabling methylation of noncanonical sites (e.g., internal adenine in AGACC) with 2–4× higher kcat on some noncanonical sequences, conferring resistance to specific host restriction systems such as EcoP1/P1, and causing virion DNA hypermethylation (Oct 2003, https://doi.org/10.1038/nsb973; Oct 1989, https://doi.org/10.1093/nar/17.20.8149) (yang2003structureofthe pages 3-4, miner1989singleaminoacid pages 1-3).
- Modern quantification in phage systems: 2024 work quantified fractions of modified adenines genome-wide using nanopore 6mA models, identified ATC as a preferred motif for a phage Dam-family MTase, and verified modifications by LC–MS/MS—illustrating current quantitative pipelines for assessing Dam-family activities in vivo (Mar 2024, https://doi.org/10.1101/2024.02.27.582347) (brandt2024discoveryofa pages 4-6).
Functional context and localization in the phage lifecycle
- Site of action and timing: T4 Dam acts in the infected E. coli cytoplasm on phage DNA, methylating GATC sites as the genome is replicated or shortly after entry/replication, providing de novo and maintenance methylation to protect DNA and influence host DNA-interacting pathways (Oct 2003, https://doi.org/10.1038/nsb973) (yang2003structureofthe pages 1-3).
- Pathway roles: By methylating GATC, T4 Dam participates in phage counter-defense against host restriction systems. Dam-mediated patterns can shield the genome from specific host endonucleases; altered specificities (e.g., damh) increase protection breadth against diverse host RM systems (Oct 2003, https://doi.org/10.1038/nsb973; Oct 1989, https://doi.org/10.1093/nar/17.20.8149) (yang2003structureofthe pages 3-4, miner1989singleaminoacid pages 1-3).
Notes on domain/family alignment
- The identified catalytic motifs (motif I, DPPY motif IV, motif VI) and the SAM-dependent fold in T4 Dam align with the UniProt-listed domains: Ade_MeTrfase_dom_2, DNA_methylase_N6_adenine consensus, and SAM-dependent_MTases superfamily (Oct 2003, https://doi.org/10.1038/nsb973) (yang2003structureofthe pages 1-3, yang2003structureofthe pages 11-15).
Conclusion
Enterobacteria phage T4 Dam (P04392) is a canonical SAM-dependent N6-adenine MTase acting at GATC sites. High-resolution structures and enzymology define its catalytic motifs, closed cofactor pocket, and processive DNA engagement. Genetic and structural evidence shows that subtle sequence changes tune specificity and anti-restriction efficacy, a critical trait for phage survival in diverse hosts. Recent 2023–2024 studies expand applications of Dam-family MTases in overcoming R–M barriers and in quantitative epigenome mapping within phage systems, underscoring the enzyme family’s ongoing utility in biotechnology and phage biology (Oct 2003, https://doi.org/10.1038/nsb973; Sep 2004, https://doi.org/10.1023/b:mbil.0000043943.07792.80; Oct 1989, https://doi.org/10.1093/nar/17.20.8149; Jul 2024, https://doi.org/10.1186/s13068-024-02529-x; Mar 2024, https://doi.org/10.1101/2024.02.27.582347; Mar 2023, https://doi.org/10.3389/fmicb.2023.1129437) (yang2003structureofthe pages 1-3, zinoviev2004molecularenzymologyof pages 1-2, miner1989singleaminoacid pages 1-3, won2024emergingmethylationbasedapproaches pages 3-5, brandt2024discoveryofa pages 4-6, gao2023bacterialdnamethyltransferase pages 3-4, gao2023bacterialdnamethyltransferase pages 13-14).
References
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(yang2003structureofthe pages 9-11): Zhe Yang, John R Horton, Lan Zhou, Xu Jia Zhang, Aiping Dong, Xing Zhang, Samuel L Schlagman, Valeri Kossykh, Stanley Hattman, and Xiaodong Cheng. Structure of the bacteriophage t4 dna adenine methyltransferase. Nature Structural Biology, 10:849-855, Oct 2003. URL: https://doi.org/10.1038/nsb973, doi:10.1038/nsb973. This article has 60 citations.
(zinoviev2004molecularenzymologyof pages 1-2): V. V. Zinoviev, A. A. Evdokimov, S. Hattman, and E. G. Malygin. Molecular enzymology of phage t4 dam dna methyltransferase. Molecular Biology, 38:737-751, Sep 2004. URL: https://doi.org/10.1023/b:mbil.0000043943.07792.80, doi:10.1023/b:mbil.0000043943.07792.80. This article has 6 citations and is from a peer-reviewed journal.
(miner1989singleaminoacid pages 1-3): Z. Miner, S. Schlagman, and Stanley Hattman. Single amino acid changes that alter the dna sequence specificity of the dna-[n6-adenine] methyltransferase (dam) of bacteriophage t4. Nucleic acids research, 17 20:8149-57, Oct 1989. URL: https://doi.org/10.1093/nar/17.20.8149, doi:10.1093/nar/17.20.8149. This article has 18 citations and is from a highest quality peer-reviewed journal.
(yang2003structureofthe pages 6-8): Zhe Yang, John R Horton, Lan Zhou, Xu Jia Zhang, Aiping Dong, Xing Zhang, Samuel L Schlagman, Valeri Kossykh, Stanley Hattman, and Xiaodong Cheng. Structure of the bacteriophage t4 dna adenine methyltransferase. Nature Structural Biology, 10:849-855, Oct 2003. URL: https://doi.org/10.1038/nsb973, doi:10.1038/nsb973. This article has 60 citations.
(yang2003structureofthe pages 3-4): Zhe Yang, John R Horton, Lan Zhou, Xu Jia Zhang, Aiping Dong, Xing Zhang, Samuel L Schlagman, Valeri Kossykh, Stanley Hattman, and Xiaodong Cheng. Structure of the bacteriophage t4 dna adenine methyltransferase. Nature Structural Biology, 10:849-855, Oct 2003. URL: https://doi.org/10.1038/nsb973, doi:10.1038/nsb973. This article has 60 citations.
(yang2003structureofthe pages 11-15): Zhe Yang, John R Horton, Lan Zhou, Xu Jia Zhang, Aiping Dong, Xing Zhang, Samuel L Schlagman, Valeri Kossykh, Stanley Hattman, and Xiaodong Cheng. Structure of the bacteriophage t4 dna adenine methyltransferase. Nature Structural Biology, 10:849-855, Oct 2003. URL: https://doi.org/10.1038/nsb973, doi:10.1038/nsb973. This article has 60 citations.
(won2024emergingmethylationbasedapproaches pages 3-5): Changhee Won and Sung Sun Yim. Emerging methylation-based approaches in microbiome engineering. Biotechnology for Biofuels and Bioproducts, Jul 2024. URL: https://doi.org/10.1186/s13068-024-02529-x, doi:10.1186/s13068-024-02529-x. This article has 6 citations and is from a domain leading peer-reviewed journal.
(brandt2024discoveryofa pages 4-6): David Brandt, Anja K. Dörrich, Marcus Persicke, Tabea Leonhard, Markus Haak, Sophia Nölting, Matthias Ruwe, Nicole Schmid, Kai M. Thormann, and Jörn Kalinowski. Discovery of a pentose as a cytosine nucleobase modification in shewanella phage thanatos-1 genomic dna mediating enhanced resistance towards host restriction systems. bioRxiv, Mar 2024. URL: https://doi.org/10.1101/2024.02.27.582347, doi:10.1101/2024.02.27.582347. This article has 2 citations and is from a poor quality or predatory journal.
(gao2023bacterialdnamethyltransferase pages 3-4): Qun Gao, Shuwei Lu, Yuwei Wang, Longgui He, Mingshu Wang, Renyong Jia, Shun Chen, Dekang Zhu, Mafeng Liu, Xinxin Zhao, Qiao Yang, Ying Wu, Shaqiu Zhang, Juan Huang, Sai Mao, Xumin Ou, Di Sun, Bin Tian, and Anchun Cheng. Bacterial dna methyltransferase: a key to the epigenetic world with lessons learned from proteobacteria. Frontiers in Microbiology, Mar 2023. URL: https://doi.org/10.3389/fmicb.2023.1129437, doi:10.3389/fmicb.2023.1129437. This article has 73 citations and is from a poor quality or predatory journal.
(gao2023bacterialdnamethyltransferase pages 13-14): Qun Gao, Shuwei Lu, Yuwei Wang, Longgui He, Mingshu Wang, Renyong Jia, Shun Chen, Dekang Zhu, Mafeng Liu, Xinxin Zhao, Qiao Yang, Ying Wu, Shaqiu Zhang, Juan Huang, Sai Mao, Xumin Ou, Di Sun, Bin Tian, and Anchun Cheng. Bacterial dna methyltransferase: a key to the epigenetic world with lessons learned from proteobacteria. Frontiers in Microbiology, Mar 2023. URL: https://doi.org/10.3389/fmicb.2023.1129437, doi:10.3389/fmicb.2023.1129437. This article has 73 citations and is from a poor quality or predatory journal.
(miner1989singleaminoacid pages 3-5): Z. Miner, S. Schlagman, and Stanley Hattman. Single amino acid changes that alter the dna sequence specificity of the dna-[n6-adenine] methyltransferase (dam) of bacteriophage t4. Nucleic acids research, 17 20:8149-57, Oct 1989. URL: https://doi.org/10.1093/nar/17.20.8149, doi:10.1093/nar/17.20.8149. This article has 18 citations and is from a highest quality peer-reviewed journal.
Reviewed the AIGR review skill, DAM-ai-review.yaml, DAM-goa.tsv, DAM-uniprot.txt, DAM-deep-research-falcon.md, and cached publications PMID_2510127.md, PMID_7782299.md, PMID_12937411.md, and PMID_15882618.md.
Core function is DNA adenine methyltransferase activity at GATC sites. The direct biochemical support is strong: [PMID:7782299 Phage T4 DNA [N6-adenine]methyltransferase. Overexpression, purification, and characterization., "T4 Dam methylates the palindromic tetranucleotide, GATC"] and [PMID:2510127 Single amino acid changes that alter the DNA sequence specificity of the DNA-[N6-adenine] methyltransferase (Dam) of bacteriophage T4., "recognizes primarily the sequence GATC in both cytosine- and hydroxymethylcytosine-containing DNA"].
The SPKW immune/defense rows are better treated as replacement cases rather than as a blanket claim that bacteria lack all defense biology. The supported mechanism is phage restriction-modification evasion by DNA methylation, not generic suppression of host innate immune response. The specific replacement target is already in GOA: GO:0099018 symbiont-mediated evasion of host restriction-modification system.
The UniProt flatfile also contains GO:1904047 S-adenosyl-L-methionine binding and GO:0006298 mismatch repair, but these are absent from the fetched DAM-goa.tsv, so they were not added to existing_annotations in this YAML pass. If those enter GOA later, SAM binding would be non-core at most; mismatch repair would need careful review because T4 Dam is a DNA modification/antirestriction enzyme, not a canonical mismatch-repair factor in this review.
id: P04392
gene_symbol: DAM
product_type: PROTEIN
status: DRAFT
taxon:
id: NCBITaxon:10665
label: Enterobacteria phage T4
description: 'T4 Dam is a DNA adenine methyltransferase (EC 2.1.1.72) that catalyzes
the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position
of adenine within GATC palindromic sequences. The enzyme functions as a monomer
and methylates both cytosine- and 5-hydroxymethylcytosine-containing DNA substrates.
The primary biological role is to protect phage T4 genomic DNA from degradation
by the host E. coli restriction-modification defense system. This is a well-characterized
"orphan" methyltransferase with high-resolution crystal structures available (PDB:
1Q0S, 1Q0T, 1YF3).'
existing_annotations:
- term:
id: GO:0003676
label: nucleic acid binding
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: T4 Dam binds DNA as part of its methyltransferase activity. Crystal structures
confirm DNA binding capability [PMID:12937411]. However, "nucleic acid binding"
is overly general for a protein with well-characterized DNA-specific binding
and enzymatic activity.
action: MODIFY
reason: This term is too general. The protein specifically binds DNA (not RNA)
in a sequence-specific manner (GATC recognition). More specific terms like GO:0003677
(DNA binding) or GO:0043565 (sequence-specific DNA binding) are more informative
and are already annotated.
proposed_replacement_terms:
- id: GO:0043565
label: sequence-specific DNA binding
supported_by:
- reference_id: PMID:12937411
supporting_text: 'T4Dam contains two domains: a seven-stranded catalytic domain
that harbors the binding site for AdoHcy and a DNA binding domain'
- reference_id: file:BPT4/DAM/DAM-deep-research-falcon.md
supporting_text: Crystal structures resolved a binary complex (Dam-AdoHcy) and
a ternary Dam-DNA-AdoHcy complex
- term:
id: GO:0003677
label: DNA binding
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: T4 Dam binds double-stranded DNA containing GATC sequences. This is well
established by crystal structures showing Dam-DNA complexes [PMID:12937411,
PMID:15882618] and biochemical studies demonstrating Km for DNA substrate of
1.1 x 10^-12 M [PMID:7782299]. The term is accurate, but it is a broad parent
of the more informative DNA methyltransferase functions.
action: KEEP_AS_NON_CORE
reason: DNA binding is necessary for Dam activity, but it is not the best core
annotation for this enzyme. The core function is site-specific DNA adenine
methyltransferase activity, with DNA binding as a supporting molecular property.
supported_by:
- reference_id: PMID:7782299
supporting_text: the Km for substrate nonglucosylated, unmethylated T4 gt- dam
DNA is 1.1 x 10(-12) M
- reference_id: PMID:12937411
supporting_text: a DNA binding domain consisting of a five-helix bundle and
a beta-hairpin
- term:
id: GO:0006260
label: DNA replication
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: While T4 Dam methylates newly replicated phage DNA, the enzyme itself
is not directly involved in DNA replication (DNA synthesis). Dam acts on DNA
after or during replication to provide methylation-based protection, but does
not participate in the replication machinery itself.
action: REMOVE
reason: T4 Dam is not part of the DNA replication machinery. It is a DNA modification
enzyme that acts on replicated DNA. The methylation protects DNA from restriction
enzymes but does not contribute to DNA synthesis. This appears to be an over-annotation
based on the UniProtKB keyword "DNA replication" which may have been applied
due to the gene's role in the phage replication cycle rather than the enzymatic
mechanism of replication.
supported_by:
- reference_id: PMID:2510127
supporting_text: Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase
(Dam)
- reference_id: file:BPT4/DAM/DAM-deep-research-falcon.md
supporting_text: T4 Dam is a DNA N6-adenine methyltransferase that transfers
a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of
adenine within GATC sites
- term:
id: GO:0008168
label: methyltransferase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: T4 Dam is indeed a methyltransferase, specifically a DNA adenine methyltransferase
(EC 2.1.1.72). This is experimentally validated [PMID:2510127, PMID:7782299].
action: KEEP_AS_NON_CORE
reason: This is accurate but quite general. The more specific term GO:0009007
(site-specific DNA-methyltransferase adenine-specific activity) is also annotated
and is more precise. Keeping this general term as a non-core parent is acceptable
because it is not incorrect, but it should not be treated as the main curated
function.
supported_by:
- reference_id: PMID:7782299
supporting_text: The bacteriophage T4 dam gene, encoding the Dam DNA [N6-adenine]methyltransferase
(MTase)
- reference_id: PMID:2510127
supporting_text: Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase
(Dam)
- term:
id: GO:0009007
label: site-specific DNA-methyltransferase (adenine-specific) activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: T4 Dam specifically methylates the N6 position of adenine within GATC
sequences. This site-specific adenine methylation activity is the core molecular
function of the enzyme, experimentally demonstrated in multiple studies [PMID:2510127,
PMID:7782299].
action: ACCEPT
reason: This is the most precise and accurate molecular function term for T4 Dam.
The enzyme recognizes the GATC palindrome and methylates the adenine at N6 position.
This is the core enzymatic function and should be retained.
supported_by:
- reference_id: PMID:7782299
supporting_text: T4 Dam methylates the palindromic tetranucleotide, GATC, designated
the canonical sequence
- reference_id: PMID:2510127
supporting_text: Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase
(Dam) which recognizes primarily the sequence GATC
- term:
id: GO:0009307
label: DNA restriction-modification system
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: T4 Dam is specifically an "orphan" methyltransferase - it is part of
a modification system but lacks a cognate restriction enzyme. The phage uses
Dam to protect its DNA FROM host restriction systems, not as part of a complete
R-M system of its own.
action: MODIFY
reason: This term implies Dam is part of a complete restriction-modification system
with both restriction and modification activities. T4 Dam is explicitly an orphan
methyltransferase (M.EcoT4Dam) that provides DNA protection without an associated
restriction enzyme. The appropriate term is GO:0099018 (symbiont-mediated evasion
of host restriction-modification system) which accurately describes the protective
role against host R-M systems.
proposed_replacement_terms:
- id: GO:0099018
label: symbiont-mediated evasion of host restriction-modification system
supported_by:
- reference_id: file:BPT4/DAM/DAM-deep-research-falcon.md
supporting_text: T4 Dam is explicitly an orphan methyltransferase (M.EcoT4Dam)
that provides DNA protection without an associated restriction enzyme
- term:
id: GO:0016740
label: transferase activity
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: T4 Dam catalyzes transfer of a methyl group from AdoMet to DNA adenine,
which is a transferase reaction. This is accurate but extremely general.
action: KEEP_AS_NON_CORE
reason: While accurate, this is a very high-level term. More specific terms (GO:0008168
methyltransferase activity, GO:0009007 site-specific DNA-methyltransferase activity)
are also present and more informative. Transferase activity is not incorrect,
but it should be retained only as a non-core parent term.
supported_by:
- reference_id: PMID:7782299
supporting_text: The bacteriophage T4 dam gene, encoding the Dam DNA [N6-adenine]methyltransferase
(MTase)
- term:
id: GO:0032259
label: methylation
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: T4 Dam catalyzes DNA adenine methylation. The enzyme is involved in the
biological process of DNA methylation to protect phage DNA.
action: MODIFY
reason: The annotation is directionally correct but too broad. T4 Dam modifies
DNA by site-specific adenine methylation; the best available non-obsolete process
replacement is GO:0006304 DNA modification, while the phage-specific outcome is
captured by GO:0099018 restriction-modification system evasion.
proposed_replacement_terms:
- id: GO:0006304
label: DNA modification
supported_by:
- reference_id: PMID:2510127
supporting_text: Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase
(Dam)
- term:
id: GO:0052031
label: symbiont-mediated perturbation of host defense response
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: This term and its definition ("A process in which a symbiont interferes
with the ability of the host to mount a defense in response to its presence")
is too broad for the specific biology. T4 Dam does not generally perturb host
defense; it methylates phage DNA so the genome can evade host restriction-
modification systems.
action: MODIFY
reason: The broad host-defense framing should be replaced by the mechanistic
phage term. The correct term for T4 Dam's role in protecting phage DNA from
bacterial restriction enzymes is GO:0099018 (symbiont-mediated evasion of host
restriction-modification system), which is already annotated and explicitly
covers phages and bacterial restriction systems.
proposed_replacement_terms:
- id: GO:0099018
label: symbiont-mediated evasion of host restriction-modification system
supported_by:
- reference_id: file:BPT4/DAM/DAM-deep-research-falcon.md
supporting_text: By methylating GATC, T4 Dam participates in phage counter-defense
against host restriction systems
- term:
id: GO:0052170
label: symbiont-mediated suppression of host innate immune response
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: This term is defined as "A process in which a symbiont inhibits or disrupts
the normal execution of the innate immune response of the host organism, the
host's first line of defense against infection." This is the wrong level of
abstraction for T4 Dam. The supported mechanism is restriction-modification
evasion by phage DNA methylation, not general suppression of an innate immune
response.
action: MODIFY
reason: This SPKW-derived annotation conflates phage antirestriction with broad
immune-response suppression. Bacteriophage T4 infects Escherichia coli, and
the evidence points to a specific bacterial restriction-modification counter-
defense mechanism. Replace this row with GO:0099018, which is already present
and accurately captures the Dam-mediated phage mechanism.
proposed_replacement_terms:
- id: GO:0099018
label: symbiont-mediated evasion of host restriction-modification system
supported_by:
- reference_id: file:BPT4/DAM/DAM-deep-research-falcon.md
supporting_text: T4 Dam acts in the infected E. coli cytoplasm on phage DNA,
methylating GATC sites as the genome is replicated
- term:
id: GO:0099018
label: symbiont-mediated evasion of host restriction-modification system
evidence_type: IEA
original_reference_id: GO_REF:0000043
review:
summary: 'This is the correct term for T4 Dam''s biological role. The GO definition
explicitly states: "A process by which a symbiont evades the DNA restriction
modification system of its host. This process occurs in phages to protect themselves
from bacterial restriction enzyme systems. Some viruses encode their own methyltransferase
in order to protect their genome from host restriction enzymes." This precisely
describes T4 Dam.'
action: ACCEPT
reason: This is the most accurate biological process term for T4 Dam. The enzyme
methylates phage DNA to protect it from E. coli restriction enzymes. The term
definition explicitly references phages, bacterial restriction systems, and
virus-encoded methyltransferases. T4 Dam is a canonical example of this mechanism.
supported_by:
- reference_id: file:BPT4/DAM/DAM-deep-research-falcon.md
supporting_text: By methylating GATC, T4 Dam participates in phage counter-defense
against host restriction systems
- reference_id: PMID:12937411
supporting_text: DNA-adenine methylation at certain GATC sites plays a pivotal
role in bacterial and phage gene expression
- term:
id: GO:0032259
label: methylation
evidence_type: IMP
original_reference_id: PMID:2510127
review:
summary: This IMP (Inferred from Mutant Phenotype) annotation is based on the
Miner et al. 1989 study that characterized dam mutants. The damh mutation (P126S)
produces a hypermethylating phenotype, demonstrating the enzyme's role in DNA
methylation in vivo.
action: MODIFY
reason: This experimental annotation is well supported, but the term itself is
broader than the biology tested. The mutant phenotype supports DNA modification
by T4 Dam, not methylation in an unrestricted substrate/process sense. Use the
more specific non-obsolete process term GO:0006304 DNA modification.
proposed_replacement_terms:
- id: GO:0006304
label: DNA modification
supported_by:
- reference_id: PMID:2510127
supporting_text: Hypermethylating mutants, damh, exhibit a relaxation in sequence
specificity, that is, they are readily able to methylate non-canonical sites
- term:
id: GO:0009008
label: DNA-methyltransferase activity
evidence_type: IDA
original_reference_id: PMID:7782299
review:
summary: This IDA (Inferred from Direct Assay) annotation is based on the Kossykh
et al. 1995 study that overexpressed, purified, and biochemically characterized
T4 Dam. The enzyme was shown to have DNA methyltransferase activity with measured
kinetic parameters.
action: ACCEPT
reason: This is high-quality experimental evidence. The study purified T4 Dam
to near homogeneity and characterized its methyltransferase activity with precise
kinetic measurements (Km for AdoMet = 0.1 uM, Km for DNA substrate = 1.1 x 10^-12
M). This directly demonstrates the core molecular function of T4 Dam.
supported_by:
- reference_id: PMID:7782299
supporting_text: 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
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings:
- statement: Provides domain-based annotations linking InterPro domains to GO terms
- id: GO_REF:0000043
title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
findings:
- statement: Source of SPKW-derived annotations including the problematic immune-related terms
- statement: Keywords like "Inhibition of host innate immune response by virus" were inappropriately mapped to phage proteins
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings:
- statement: Combined automated annotation pipeline
- id: PMID:2510127
title: Single amino acid changes that alter the DNA sequence specificity of the DNA-[N6-adenine]
methyltransferase (Dam) of bacteriophage T4.
findings:
- statement: Characterized damh hypermethylating mutant (P126S)
supporting_text: "Hypermethylating mutants, damh, exhibit a relaxation in sequence specificity"
- statement: Demonstrated T4 Dam recognizes GATC in both cytosine and hydroxymethylcytosine DNA
supporting_text: "Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam) which recognizes primarily the sequence GATC in both cytosine- and hydroxymethylcytosine-containing DNA"
- statement: Identified homology region III as involved in DNA sequence recognition
supporting_text: "These results implicate homology region III as a domain involved in DNA sequence recognition"
- statement: damc mutant (F127V) methylates GATC in cytosine-DNA but not hmC-DNA
supporting_text: "damc, which methylates GATC in cytosine-containing DNA, but not in hydroxymethylcytosine-containing DNA"
- id: PMID:7782299
title: Phage T4 DNA [N6-adenine]methyltransferase. Overexpression, purification, and
characterization.
findings:
- statement: Purified T4 Dam to near homogeneity
supporting_text: "was developed to purify the enzyme to near homogeneity"
- statement: Determined enzyme is a monomer (s20,w = 3.0 S, Stokes radius = 23 A)
supporting_text: "The MTase has an s20,w of 3.0 S and a Stokes radius of 23 A and exists in solution as a monomer"
- statement: Measured Km for AdoMet = 0.1 uM
supporting_text: "The Km for the methyl donor, S-adenosylmethionine, is 0.1 x 10(-6) M"
- statement: Measured Km for DNA substrate = 1.1 x 10^-12 M
supporting_text: "the Km for substrate nonglucosylated, unmethylated T4 gt- dam DNA is 1.1 x 10(-12) M"
- statement: Products (AdoHcy and methylated DNA) are competitive inhibitors
supporting_text: "The products of DNA methylation, S-adenosyl-L-homocysteine and methylated DNA, are competitive inhibitors of the reaction"
- statement: T4 Dam methylates canonical GATC; can methylate GAY at high enzyme:DNA ratios
supporting_text: "T4 Dam methylates the palindromic tetranucleotide, GATC, designated the canonical sequence. However, at high MTase:DNA ratios, T4 Dam can methylate some noncanonical sequences"
- id: PMID:12654995
title: A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases
and their genes.
findings:
- statement: Provides nomenclature system for restriction enzymes and methyltransferases
supporting_text: "A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes"
- id: PMID:12937411
title: Structure of the bacteriophage T4 DNA adenine methyltransferase.
findings:
- statement: Solved crystal structure of T4 Dam in binary and ternary complexes
supporting_text: "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"
- statement: Confirmed two-domain structure with catalytic and DNA binding domains
supporting_text: "T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain"
- statement: Two Dam monomers bind per DNA duplex in nonspecific mode
supporting_text: "the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex"
- id: PMID:15882618
title: Transition from nonspecific to specific DNA interactions along the substrate-recognition
pathway of dam methyltransferase.
findings:
- statement: Characterized DNA recognition mechanism of Dam methyltransferase
supporting_text: "Three structures are described for bacteriophage T4 DNA-adenine methyltransferase (T4Dam) in ternary complexes with partially and fully specific DNA and a methyl-donor analog...We have identified two types of protein-DNA interactions: discriminatory contacts, which stabilize the transition state and accelerate methylation of the cognate site, and antidiscriminatory contacts"
- id: file:BPT4/DAM/DAM-deep-research-falcon.md
title: Deep research summary for T4 Dam
findings:
- statement: T4 Dam is an orphan methyltransferase without cognate restriction enzyme
supporting_text: "T4 Dam is explicitly an orphan methyltransferase (M.EcoT4Dam) that provides DNA protection without an associated restriction enzyme"
- statement: Dam methylation protects phage DNA from host restriction systems
supporting_text: "By methylating GATC, T4 Dam participates in phage counter-defense against host restriction systems"
core_functions:
- molecular_function:
id: GO:0009007
label: site-specific DNA-methyltransferase (adenine-specific) activity
description: T4 Dam catalyzes the transfer of a methyl group from S-adenosyl-L-methionine
to the N6 position of adenine within GATC sequences. This is the core molecular
function of the enzyme, demonstrated by direct biochemical assays [PMID:7782299]
and structural studies [PMID:12937411]. The enzyme has high affinity for both
cofactor (Km = 0.1 uM for AdoMet) and substrate DNA (Km = 1.1 x 10^-12 M).
directly_involved_in:
- id: GO:0099018
label: symbiont-mediated evasion of host restriction-modification system
supported_by:
- reference_id: PMID:7782299
supporting_text: T4 Dam methylates the palindromic tetranucleotide, GATC, designated
the canonical sequence
- reference_id: file:BPT4/DAM/DAM-deep-research-falcon.md
supporting_text: By methylating GATC, T4 Dam participates in phage counter-defense
against host restriction systems
proposed_new_terms: []
suggested_questions:
- question: What is the precise timing of Dam methylation during phage infection -
does it occur co-replicationally or post-replicationally?
- question: Are there E. coli restriction systems that T4 specifically evades through
Dam methylation, and which host strains show differential susceptibility to dam-
phage?
suggested_experiments:
- description: Systematic analysis of T4 dam- phage viability across E. coli strains
with different restriction-modification systems to catalog the protective scope
of Dam methylation.
hypothesis: T4 Dam methylation provides selective protection against specific host
restriction systems, and dam- phage will show differential viability depending
on the host R-M system present.