TY - JOUR
T1 - Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation
AU - Hashimoto, Hideharu
AU - Liu, Yiwei
AU - Upadhyay, Anup K.
AU - Chang, Yanqi
AU - Howerton, Shelley B.
AU - Vertino, Paula M.
AU - Zhang, Xing
AU - Cheng, Xiaodong
PY - 2012/6
Y1 - 2012/6
N2 - Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5methylcytosine (M; 5mC) and 5hydroxymethylcytosine (H; 5hmC). During semiconservative DNA replication, hemi-methylated (M/C) and hemi- hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.
AB - Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5methylcytosine (M; 5mC) and 5hydroxymethylcytosine (H; 5hmC). During semiconservative DNA replication, hemi-methylated (M/C) and hemi- hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.
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U2 - 10.1093/nar/gks155
DO - 10.1093/nar/gks155
M3 - Article
C2 - 22362737
AN - SCOPUS:84860221291
SN - 0305-1048
VL - 40
SP - 4841
EP - 4849
JO - Nucleic acids research
JF - Nucleic acids research
IS - 11
ER -