Human HemK2/KMT9/N6AMT1 is an active protein methyltransferase, but does not act on DNA in vitro, in the presence of Trm112

Woodcock, Clayton B.; Yu, Dan; Zhang, Xing; Cheng, Xiaodong

doi:10.1038/s41421-019-0119-5

Cited by 40 publications

(44 citation statements)

References 15 publications

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“…However, direct activity of the enzyme (in complex with partner protein Trm112) on DNA was subsequently excluded. [ 45 ] Other methyltransferases that have been implicated in adenine DNA methylation belong to the METTL4/DAMT1 family of methyltransferases, that is eukaryotic subclade 3 of the MT‐A70 clade of the group I methyltransferases. [ 14 ] This places the candidate methyltransferases in a separate eukaryotic subclade, within the MT‐A70 clade that also harbors the adenine DNA methyltransferases of ciliates and algae.…”

Section: Ma In the Dna Of Animals And Plantsmentioning

confidence: 99%

DNA adenine methylation in eukaryotes: Enzymatic mark or a form of DNA damage?

Bochtler

Fernandes

2020

BioEssays

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6‐methyladenine (6mA) is fairly abundant in nuclear DNA of basal fungi, ciliates and green algae. In these organisms, 6mA is maintained near transcription start sites in ApT context by a parental‐strand instruction dependent maintenance methyltransferase and is positively associated with transcription. In animals and plants, 6mA levels are high only in organellar DNA. The 6mA levels in nuclear DNA are very low. They are attributable to nucleotide salvage and the activity of otherwise mitochondrial METTL4, and may be considered as a price that cells pay for adenine methylation in RNA and/or organellar DNA. Cells minimize this price by sanitizing dNTP pools to limit 6mA incorporation, and by converting 6mA that has been incorporated into DNA back to adenine. Hence, 6mA in nuclear DNA should be described as an epigenetic mark only in basal fungi, ciliates and green algae, but not in animals and plants.

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Section: Ma In the Dna Of Animals And Plantsmentioning

confidence: 99%

DNA adenine methylation in eukaryotes: Enzymatic mark or a form of DNA damage?

Bochtler

Fernandes

2020

BioEssays

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Section: Introductionmentioning

confidence: 99%

“…Second, there is also uncertainty regarding the mammalian enzyme(s) (suggested to include HemK2/N6AMA1, MettL3-MettL14 complex and MettL4) responsible for generating N6mA in mammalian DNA ( 10 , 13 , 19–21 ). Human HemK2, which was thought to be a DNA N6mA MTase ( 11 ), is actually a protein MTase active on glutamine and lysine ( 19 , 20 , 22 , 23 ). Guided by the sequential order of conserved sequence motifs unique to Class β amino (N6mA and N4mC) MTases ( 4 , 24 , 25 ), we identified human MettL3–MettL14 as a DNA adenine MTase active on single-strand and unpaired DNA in vitro ( 26 ).…”

Section: Introductionmentioning

confidence: 99%

Biochemical and structural basis for YTH domain of human YTHDC1 binding to methylated adenine in DNA

Woodcock

Horton

Zhou

et al. 2020

Nucleic Acids Research

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The recently characterized mammalian writer (methyltransferase) and eraser (demethylase) of the DNA N6-methyladenine (N6mA) methyl mark act on single-stranded (ss) and transiently-unpaired DNA. As YTH domain-containing proteins bind N6mA-containing RNA in mammalian cells, we investigated whether mammalian YTH domains are also methyl mark readers of N6mA DNA. Here, we show that the YTH domain of YTHDC1 (known to localize in the nucleus) binds ssDNA containing N6mA, with a 10 nM dissociation constant. This binding is stronger by a factor of 5 than in an RNA context, tested under the same conditions. However, the YTH domains of YTHDF2 and YTHDF1 (predominantly cytoplasmic) exhibited the opposite effect with ∼1.5–2× stronger binding to ssRNA containing N6mA than to the corresponding DNA. We determined two structures of the YTH domain of YTHDC1 in complex with N6mA-containing ssDNA, which illustrated that YTHDC1 binds the methylated adenine in a single-stranded region flanked by duplexed DNA. We discuss the hypothesis that the writer-reader-eraser of N6mA-containining ssDNA is associated with maintaining genome stability. Structural comparison of YTH and SRA domains (the latter a DNA 5-methylcytosine reader) revealed them to be diverse members of a larger family of DNA/RNA modification readers, apparently having originated from bacterial modification-dependent restriction enzymes.

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“…SETD3 has optimal pH at ≥ 7 for histidine methylation, but 10.5 for lysine methylation ( 58 ). HemK2-Trm112 complex is most active for glutamine methylation at pH 8.0 but for lysine methylation at pH 10.5 ( 15 ). In this study, PCIF1 showed higher activity on mRNA cap analog at low (5.4) and high pH (9.4) than at intermediate pH levels.…”

Section: Discussionmentioning

confidence: 99%

“…While noted immunochemically as early as 1983 ( 8 ), N6mA was reported in mammalian DNA using very sensitive approaches only in 2016 ( 9 ), and its existence in mammals is still debatable ( 10 , 11 ). Remaining unsettled questions include how N6mA is generated in mammalian DNA ( 12 , 13 ) and identification of the potential adenine DNA MTase(s) ( 14 , 15 , 16 ).…”

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confidence: 99%

Enzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima

Kaur

Blumenthal

et al. 2021

Journal of Biological Chemistry

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RNA methylations of varied RNA species (mRNA, tRNA, rRNA, non-coding RNA) generate a range of modified nucleotides, including N6-methyladenosine. Here we study the enzymology of three human RNA methyltransferases that methylate the adenosine amino group in diverse contexts, when it is: the first transcribed nucleotide after the mRNA cap (PCIF1), at position 1832 of 18S rRNA (MettL5-Trm112 complex), and within a hairpin in the 3′ UTR of the S-adenosyl- l -methionine synthetase (MettL16). Among these three enzymes, the catalytic efficiency ranges from PCIF1, with the fastest turnover rate of >230 h −1 μM −1 on mRNA cap analog, down to MettL16, which has the lowest rate of ∼3 h −1 μM −1 acting on an RNA hairpin. Both PCIF1 and MettL5 have a binding affinity ( K m ) of ∼1 μM or less for both substrates of SAM and RNA, whereas MettL16 has significantly lower binding affinities for both ( K m >0.4 mM for SAM and ∼10 μM for RNA). The three enzymes are active over a wide pH range (∼5.4–9.4) and have different preferences for ionic strength. Sodium chloride at 200 mM markedly diminished methylation activity of MettL5-Trm112 complex, whereas MettL16 had higher activity in the range of 200 to 500 mM NaCl. Zinc ion inhibited activities of all three enzymes. Together, these results illustrate the diversity of RNA adenosine methyltransferases in their enzymatic mechanisms and substrate specificities and underline the need for assay optimization in their study.

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Human HemK2/KMT9/N6AMT1 is an active protein methyltransferase, but does not act on DNA in vitro, in the presence of Trm112

Cited by 40 publications

References 15 publications

DNA adenine methylation in eukaryotes: Enzymatic mark or a form of DNA damage?

DNA adenine methylation in eukaryotes: Enzymatic mark or a form of DNA damage?

Biochemical and structural basis for YTH domain of human YTHDC1 binding to methylated adenine in DNA

Enzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima

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