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Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.

Hashimoto H, Pais JE, Zhang X, Saleh L, Fu ZQ, Dai N, Corrêa IR, Zheng Y, Cheng X - Nature (2013)

Bottom Line: Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1.The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC.The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA.

ABSTRACT
Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

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Activity of NgTet1a, Detection of 5hmC (top) and 5caC (bottom) by antibodies. b, The relative amount of each reaction product was sequentially observed over the full time course of the reaction. c, NgTet1 is active on all three DNA substrates, producing 5caC. d, Quantitative LC-MS measurement of 5mC disappearance and formation of 5hmC, 5fC and 5caC. e, The effects of mutations on the conversion of 5mC. Error bars indicate s.d. of the mean value from three independent experiments.
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Figure 1: Activity of NgTet1a, Detection of 5hmC (top) and 5caC (bottom) by antibodies. b, The relative amount of each reaction product was sequentially observed over the full time course of the reaction. c, NgTet1 is active on all three DNA substrates, producing 5caC. d, Quantitative LC-MS measurement of 5mC disappearance and formation of 5hmC, 5fC and 5caC. e, The effects of mutations on the conversion of 5mC. Error bars indicate s.d. of the mean value from three independent experiments.

Mentions: The free-living amoeboflagellate Naegleria gruberi has eight Tet/JBP-like dioxygenases (NgTet1-8; Extended Data Fig. 1). The NgTet proteins vary in length, but all contain a conserved core region of ~210 residues including the invariant Fe(II)-binding histidines and aspartate (the HxD…H motif). We measured NgTet1 activity using various double-stranded DNA as substrates, each containing a single modified base X within a G:X pair in a CpG sequence. We used antibodies specific for 5hmC, 5fC and 5caC (Extended Data Fig. 2a–c). Using 5mC-containing DNA as substrate, 5hmC (the first reaction product) and 5caC (the last reaction product) are detected in the presence of α-ketoglutarate (αKG), but not with N-oxalylglycine (NOG) (Fig. 1a). NgTet1 initially produces 5hmC at 5min, 5fC between 5 to 10min and finally 5caC at 15min under the assay conditions (Fig. 1b). NgTet1 is active on all three DNA substrates containing 5mC, 5hmC or 5fC, generating 5caC (Fig. 1c). We applied quantitative mass spectrometry to monitor the kinetics of product formation (Fig. 1d and Extended Data Fig. 2d). When the amount of 5mC rapidly disappears (2–5min), a peak of 5hmC forms transiently before being converted to 5fC and 5caC products (Fig. 1d). The first conversion from 5mC to 5hmC is faster (kobs=21h−1) than the second conversion from 5hmC (kobs≈3h−1). In addition, we used human thymine DNA glycosylase to probe the products generated by NgTet1 (Extended Data Fig. 2e).


Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.

Hashimoto H, Pais JE, Zhang X, Saleh L, Fu ZQ, Dai N, Corrêa IR, Zheng Y, Cheng X - Nature (2013)

Activity of NgTet1a, Detection of 5hmC (top) and 5caC (bottom) by antibodies. b, The relative amount of each reaction product was sequentially observed over the full time course of the reaction. c, NgTet1 is active on all three DNA substrates, producing 5caC. d, Quantitative LC-MS measurement of 5mC disappearance and formation of 5hmC, 5fC and 5caC. e, The effects of mutations on the conversion of 5mC. Error bars indicate s.d. of the mean value from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4364404&req=5

Figure 1: Activity of NgTet1a, Detection of 5hmC (top) and 5caC (bottom) by antibodies. b, The relative amount of each reaction product was sequentially observed over the full time course of the reaction. c, NgTet1 is active on all three DNA substrates, producing 5caC. d, Quantitative LC-MS measurement of 5mC disappearance and formation of 5hmC, 5fC and 5caC. e, The effects of mutations on the conversion of 5mC. Error bars indicate s.d. of the mean value from three independent experiments.
Mentions: The free-living amoeboflagellate Naegleria gruberi has eight Tet/JBP-like dioxygenases (NgTet1-8; Extended Data Fig. 1). The NgTet proteins vary in length, but all contain a conserved core region of ~210 residues including the invariant Fe(II)-binding histidines and aspartate (the HxD…H motif). We measured NgTet1 activity using various double-stranded DNA as substrates, each containing a single modified base X within a G:X pair in a CpG sequence. We used antibodies specific for 5hmC, 5fC and 5caC (Extended Data Fig. 2a–c). Using 5mC-containing DNA as substrate, 5hmC (the first reaction product) and 5caC (the last reaction product) are detected in the presence of α-ketoglutarate (αKG), but not with N-oxalylglycine (NOG) (Fig. 1a). NgTet1 initially produces 5hmC at 5min, 5fC between 5 to 10min and finally 5caC at 15min under the assay conditions (Fig. 1b). NgTet1 is active on all three DNA substrates containing 5mC, 5hmC or 5fC, generating 5caC (Fig. 1c). We applied quantitative mass spectrometry to monitor the kinetics of product formation (Fig. 1d and Extended Data Fig. 2d). When the amount of 5mC rapidly disappears (2–5min), a peak of 5hmC forms transiently before being converted to 5fC and 5caC products (Fig. 1d). The first conversion from 5mC to 5hmC is faster (kobs=21h−1) than the second conversion from 5hmC (kobs≈3h−1). In addition, we used human thymine DNA glycosylase to probe the products generated by NgTet1 (Extended Data Fig. 2e).

Bottom Line: Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1.The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC.The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA.

ABSTRACT
Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

Show MeSH
Related in: MedlinePlus