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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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Related in: MedlinePlus

DNA synthesis reactions of human Polδ on DNA templates containing 5caC.(A) 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 (A) with increasing amounts of Polδ (0, 0.8, and 4 nM in each group of three lanes) at 30°C for 5 min. (B and C) A 17-mer primer was 5′-labeled with 32P and annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). (B) The primer/template complexes were incubated with Polδ exo- (10 nM) for 5 min at 30°C with one of the indicated dNTPs (lanes 1–4, 5–8, 9–12, 13–16, and 17–20). (C) The 5′-labeled 17-mer primer was loading in the first lane. The primer/template complexes were incubated with pol δ (4 nM) and dGTP (100 μM) at 30°C for 5 min.
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f2: DNA synthesis reactions of human Polδ on DNA templates containing 5caC.(A) 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 (A) with increasing amounts of Polδ (0, 0.8, and 4 nM in each group of three lanes) at 30°C for 5 min. (B and C) A 17-mer primer was 5′-labeled with 32P and annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). (B) The primer/template complexes were incubated with Polδ exo- (10 nM) for 5 min at 30°C with one of the indicated dNTPs (lanes 1–4, 5–8, 9–12, 13–16, and 17–20). (C) The 5′-labeled 17-mer primer was loading in the first lane. The primer/template complexes were incubated with pol δ (4 nM) and dGTP (100 μM) at 30°C for 5 min.

Mentions: Both KF exo- and Polη were incapable of proofreading during DNA synthesis; therefore, we investigated whether human DNA polymerase δ (Polδ), which harbors an intrinsic 3′ to 5′ exonuclease domain, catalyzes DNA synthesis past the modified cytosines during replication. Interestingly, although Polδ synthesized DNA fragments on all templates, proofreading cleavage products were observed with only the 5caC templates (Figure 2A). Next, we examined the nucleotide preference for incorporation opposite a modified cytosine by Polδ exo-, which catalyzes DNA synthesis (Figure S2) and lacks 3′ to 5′ exonuclease activity. As shown in Figure 2B, this polymerase also incorporated dGTP opposite modified cytosines.


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 reactions of human Polδ on DNA templates containing 5caC.(A) 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 (A) with increasing amounts of Polδ (0, 0.8, and 4 nM in each group of three lanes) at 30°C for 5 min. (B and C) A 17-mer primer was 5′-labeled with 32P and annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). (B) The primer/template complexes were incubated with Polδ exo- (10 nM) for 5 min at 30°C with one of the indicated dNTPs (lanes 1–4, 5–8, 9–12, 13–16, and 17–20). (C) The 5′-labeled 17-mer primer was loading in the first lane. The primer/template complexes were incubated with pol δ (4 nM) and dGTP (100 μM) at 30°C for 5 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: DNA synthesis reactions of human Polδ on DNA templates containing 5caC.(A) 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 (A) with increasing amounts of Polδ (0, 0.8, and 4 nM in each group of three lanes) at 30°C for 5 min. (B and C) A 17-mer primer was 5′-labeled with 32P and annealed with a 30-mer oligonucleotide containing the indicated modified cytosines at position X (upper panel). (B) The primer/template complexes were incubated with Polδ exo- (10 nM) for 5 min at 30°C with one of the indicated dNTPs (lanes 1–4, 5–8, 9–12, 13–16, and 17–20). (C) The 5′-labeled 17-mer primer was loading in the first lane. The primer/template complexes were incubated with pol δ (4 nM) and dGTP (100 μM) at 30°C for 5 min.
Mentions: Both KF exo- and Polη were incapable of proofreading during DNA synthesis; therefore, we investigated whether human DNA polymerase δ (Polδ), which harbors an intrinsic 3′ to 5′ exonuclease domain, catalyzes DNA synthesis past the modified cytosines during replication. Interestingly, although Polδ synthesized DNA fragments on all templates, proofreading cleavage products were observed with only the 5caC templates (Figure 2A). Next, we examined the nucleotide preference for incorporation opposite a modified cytosine by Polδ exo-, which catalyzes DNA synthesis (Figure S2) and lacks 3′ to 5′ exonuclease activity. As shown in Figure 2B, this polymerase also incorporated dGTP opposite modified cytosines.

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