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Guanine- 5-carboxylcytosine base pairs mimic mismatches during DNA replication.

Shibutani T, Ito S, Toda M, Kanao R, Collins LB, Shibata M, Urabe M, Koseki H, Masuda Y, Swenberg JA, Masutani C, Hanaoka F, Iwai S, Kuraoka I - Sci Rep (2014)

Bottom Line: The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity.Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC.These results suggest the epigenetic modification products of 5caC behave as DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Engineering Science, Osaka University Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531 Japan.

ABSTRACT
The genetic information encoded in genomes must be faithfully replicated and transmitted to daughter cells. The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity. Here, we have shown that although 5caC pairs with guanine during DNA replication in vitro, G·5caC pairs stimulated DNA polymerase exonuclease activity and were recognized by the mismatch repair (MMR) proteins. Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC. These results suggest the epigenetic modification products of 5caC behave as DNA lesions.

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DNA synthesis by Klenow fragment exonuclease minus (KF exo-) on a DNA template containing 5fC and 5caC.(A) Proposed 5mC oxidation pathway involving TET dioxygenases. 5fC and 5caC are removed by TDG in the BER pathway. (B) A 13-mer primer was 5′-labeled with 32P and annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). The primer/template complexes were incubated with increasing amounts of KF exo- (0, 0.04, and 1 U in each group of three lanes) at 30°C for 5 min. (C) Relative KF exo- DNA synthesis efficiency on all 5 modified cytosine templates. Data were normalized to the DNA synthesis efficiency of KF exo- (1 U) for normal cytosine-containing templates (lane 3). Quantification of the 30–31-nt fragments by image analysis. Error bars indicate the standard deviation. n.d.: not determined. (D) A 17-mer primer was 5′-labeled with 32P and was annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). The primer/template complexes were incubated with KF exo- (0.01 U) for 5 min on ice with one of the indicated dNTPs (lanes 1–4, 5–8, 9–12, 13–16, and 17–20).
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f1: DNA synthesis by Klenow fragment exonuclease minus (KF exo-) on a DNA template containing 5fC and 5caC.(A) Proposed 5mC oxidation pathway involving TET dioxygenases. 5fC and 5caC are removed by TDG in the BER pathway. (B) A 13-mer primer was 5′-labeled with 32P and annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). The primer/template complexes were incubated with increasing amounts of KF exo- (0, 0.04, and 1 U in each group of three lanes) at 30°C for 5 min. (C) Relative KF exo- DNA synthesis efficiency on all 5 modified cytosine templates. Data were normalized to the DNA synthesis efficiency of KF exo- (1 U) for normal cytosine-containing templates (lane 3). Quantification of the 30–31-nt fragments by image analysis. Error bars indicate the standard deviation. n.d.: not determined. (D) A 17-mer primer was 5′-labeled with 32P and was annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). The primer/template complexes were incubated with KF exo- (0.01 U) for 5 min on ice with one of the indicated dNTPs (lanes 1–4, 5–8, 9–12, 13–16, and 17–20).

Mentions: During DNA replication, maintenance DNMT1 maintains symmetric CpG methylation with high specificity on the unmethylated strand of a hemi-methylated CpG sequence, but not in a hemi-hydroxymethylated CpG sequence, which could lead to passive DNA methylation1617. Alternatively, 5hmC can be converted to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) via TET protein-mediated consecutive oxidations (Figure 1A)1819. Biochemical analyses suggest thymine DNA glycosylase (TDG) excises 5fC and 5caC, thereby generating an apurinic/apyrimidinic site that is in turn processed by the base excision repair (BER) machinery, suggesting an active DNA demethylation pathway20. The importance of TDG in maintaining appropriate DNA methylation has been indicated by targeted Tdg allele disruptions in mice; these knockout mice exhibit aberrant DNA methylation in a subset of gene promoters and enhancers, coincident with dysregulated gene expression2122. Unlike other DNA glycosylases required for the BER pathway, Tdg knockout is embryonic lethal in mice, despite leaving DNA repair largely intact. Thus, TDG is essential for proper embryonic development, in part due to its role in maintaining epigenetic stability during cell-lineage commitment.


Guanine- 5-carboxylcytosine base pairs mimic mismatches during DNA replication.

Shibutani T, Ito S, Toda M, Kanao R, Collins LB, Shibata M, Urabe M, Koseki H, Masuda Y, Swenberg JA, Masutani C, Hanaoka F, Iwai S, Kuraoka I - Sci Rep (2014)

DNA synthesis by Klenow fragment exonuclease minus (KF exo-) on a DNA template containing 5fC and 5caC.(A) Proposed 5mC oxidation pathway involving TET dioxygenases. 5fC and 5caC are removed by TDG in the BER pathway. (B) A 13-mer primer was 5′-labeled with 32P and annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). The primer/template complexes were incubated with increasing amounts of KF exo- (0, 0.04, and 1 U in each group of three lanes) at 30°C for 5 min. (C) Relative KF exo- DNA synthesis efficiency on all 5 modified cytosine templates. Data were normalized to the DNA synthesis efficiency of KF exo- (1 U) for normal cytosine-containing templates (lane 3). Quantification of the 30–31-nt fragments by image analysis. Error bars indicate the standard deviation. n.d.: not determined. (D) A 17-mer primer was 5′-labeled with 32P and was annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). The primer/template complexes were incubated with KF exo- (0.01 U) for 5 min on ice with one of the indicated dNTPs (lanes 1–4, 5–8, 9–12, 13–16, and 17–20).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048885&req=5

f1: DNA synthesis by Klenow fragment exonuclease minus (KF exo-) on a DNA template containing 5fC and 5caC.(A) Proposed 5mC oxidation pathway involving TET dioxygenases. 5fC and 5caC are removed by TDG in the BER pathway. (B) A 13-mer primer was 5′-labeled with 32P and annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). The primer/template complexes were incubated with increasing amounts of KF exo- (0, 0.04, and 1 U in each group of three lanes) at 30°C for 5 min. (C) Relative KF exo- DNA synthesis efficiency on all 5 modified cytosine templates. Data were normalized to the DNA synthesis efficiency of KF exo- (1 U) for normal cytosine-containing templates (lane 3). Quantification of the 30–31-nt fragments by image analysis. Error bars indicate the standard deviation. n.d.: not determined. (D) A 17-mer primer was 5′-labeled with 32P and was annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). The primer/template complexes were incubated with KF exo- (0.01 U) for 5 min on ice with one of the indicated dNTPs (lanes 1–4, 5–8, 9–12, 13–16, and 17–20).
Mentions: During DNA replication, maintenance DNMT1 maintains symmetric CpG methylation with high specificity on the unmethylated strand of a hemi-methylated CpG sequence, but not in a hemi-hydroxymethylated CpG sequence, which could lead to passive DNA methylation1617. Alternatively, 5hmC can be converted to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) via TET protein-mediated consecutive oxidations (Figure 1A)1819. Biochemical analyses suggest thymine DNA glycosylase (TDG) excises 5fC and 5caC, thereby generating an apurinic/apyrimidinic site that is in turn processed by the base excision repair (BER) machinery, suggesting an active DNA demethylation pathway20. The importance of TDG in maintaining appropriate DNA methylation has been indicated by targeted Tdg allele disruptions in mice; these knockout mice exhibit aberrant DNA methylation in a subset of gene promoters and enhancers, coincident with dysregulated gene expression2122. Unlike other DNA glycosylases required for the BER pathway, Tdg knockout is embryonic lethal in mice, despite leaving DNA repair largely intact. Thus, TDG is essential for proper embryonic development, in part due to its role in maintaining epigenetic stability during cell-lineage commitment.

Bottom Line: The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity.Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC.These results suggest the epigenetic modification products of 5caC behave as DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Engineering Science, Osaka University Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531 Japan.

ABSTRACT
The genetic information encoded in genomes must be faithfully replicated and transmitted to daughter cells. The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity. Here, we have shown that although 5caC pairs with guanine during DNA replication in vitro, G·5caC pairs stimulated DNA polymerase exonuclease activity and were recognized by the mismatch repair (MMR) proteins. Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC. These results suggest the epigenetic modification products of 5caC behave as DNA lesions.

Show MeSH
Related in: MedlinePlus