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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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MutSα complex binds to G·5caC base pairs.(A) Postulated base-pairing models of 5caC with G. (B and C) A 34-mer oligonucleotide was 5′-labeled with 32P and annealed with a 34-mer oligonucleotide containing the 5caC. The mismatch substrates were incubated (B) with MutSα on ice for 20 min or (C) with MutSα and cold G·T mismatch substrates (lanes 3–6: non-labeled substrates/5′-labeled substrates molar ratio; ×1, ×10, ×50, and ×100) at 25°C for 20 min. Free and bound fractions were separated on nondenaturing 6% polyacrylamide gels containing 5 μM MgCl2. (D and E) Biotin-labeled 34-mer mismatch substrates were incubated (D) with whole cell extracts (10 μg) or (F) with whole cell extracts (10 μg) and G·T mismatch substrates (lanes 3–5: non-labeled substrates/5′-labeled substrates molar ratio; ×1, ×10, and ×50). Samples were loaded on 10% SDS-page gels, and MSH2 and MSH6 were detected by western blotting.
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f3: MutSα complex binds to G·5caC base pairs.(A) Postulated base-pairing models of 5caC with G. (B and C) A 34-mer oligonucleotide was 5′-labeled with 32P and annealed with a 34-mer oligonucleotide containing the 5caC. The mismatch substrates were incubated (B) with MutSα on ice for 20 min or (C) with MutSα and cold G·T mismatch substrates (lanes 3–6: non-labeled substrates/5′-labeled substrates molar ratio; ×1, ×10, ×50, and ×100) at 25°C for 20 min. Free and bound fractions were separated on nondenaturing 6% polyacrylamide gels containing 5 μM MgCl2. (D and E) Biotin-labeled 34-mer mismatch substrates were incubated (D) with whole cell extracts (10 μg) or (F) with whole cell extracts (10 μg) and G·T mismatch substrates (lanes 3–5: non-labeled substrates/5′-labeled substrates molar ratio; ×1, ×10, and ×50). Samples were loaded on 10% SDS-page gels, and MSH2 and MSH6 were detected by western blotting.

Mentions: The proofreading function of DNA polymerases plays an important role in correcting replicative mismatch errors. Our results suggest this proofreading occurs at G·5caC pairings but not at other cytosine pairings. Although 5caC forms appropriate base pairs with guanine, we hypothesized that these pairings behave like mismatches (Figure 3A). If this holds true, the mismatch repair (MMR) protein MutS should recognize both pairings as it does G·T mismatches, which are a canonical MutS substrate313233. To test this possibility, we performed electrophoretic mobility shift assays (EMSAs) with Taq MutS and 30-mer DNA substrates containing G·T, G·C, G·5mC, G·5hmC, G·5fC, and G·5caC. We observed a striking difference in MutS binding efficiency between these forms of cytosine. MutS bound G·T and G·5caC pairs (Figure S4A and S4B). The binding preference order was G·T = G·5caC > G·5fC > G·C = G·5mC = G·5hmC. Next, we performed EMSAs with 34-mer G·5caC-containing DNA substrates and human MMR protein MutSα complexes, which consist of MSH2 and MSH6 (Figure S5), because the exonuclease activity of Polδ was observed only on the 5caC templates. The MutSα complex is a human homolog of the MMR protein MutS and is indispensable for the mammalian MMR system3435. MutSα bound to the positive control G·T pairs and to the G·5caC pairs (Figure 3B); addition of excess cold G·T DNA substrates inhibited binding between MutSα and G·5caC DNA substrates (Figure 3C). To confirm this interaction, biotin labeled-G·5caC DNA substrates were incubated with HeLa whole cell extracts and the DNA-bound proteins were pulled down with streptavidin-coated beads; MSH2 and MSH6 were detected by immunoblotting. Results confirmed the MutSα complex recognized G·5caC pairs in DNA substrates (Figures 3D and 3E). Thus, the G·5caC pairs behaved similarly to a G·T mismatch when Polδ synthesized new DNA fragments opposite 5caC, although DNA polymerase correctly incorporated dGTP. In addition, G·5caC pairs may be subjected to MMR in mammalian cells.


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)

MutSα complex binds to G·5caC base pairs.(A) Postulated base-pairing models of 5caC with G. (B and C) A 34-mer oligonucleotide was 5′-labeled with 32P and annealed with a 34-mer oligonucleotide containing the 5caC. The mismatch substrates were incubated (B) with MutSα on ice for 20 min or (C) with MutSα and cold G·T mismatch substrates (lanes 3–6: non-labeled substrates/5′-labeled substrates molar ratio; ×1, ×10, ×50, and ×100) at 25°C for 20 min. Free and bound fractions were separated on nondenaturing 6% polyacrylamide gels containing 5 μM MgCl2. (D and E) Biotin-labeled 34-mer mismatch substrates were incubated (D) with whole cell extracts (10 μg) or (F) with whole cell extracts (10 μg) and G·T mismatch substrates (lanes 3–5: non-labeled substrates/5′-labeled substrates molar ratio; ×1, ×10, and ×50). Samples were loaded on 10% SDS-page gels, and MSH2 and MSH6 were detected by western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048885&req=5

f3: MutSα complex binds to G·5caC base pairs.(A) Postulated base-pairing models of 5caC with G. (B and C) A 34-mer oligonucleotide was 5′-labeled with 32P and annealed with a 34-mer oligonucleotide containing the 5caC. The mismatch substrates were incubated (B) with MutSα on ice for 20 min or (C) with MutSα and cold G·T mismatch substrates (lanes 3–6: non-labeled substrates/5′-labeled substrates molar ratio; ×1, ×10, ×50, and ×100) at 25°C for 20 min. Free and bound fractions were separated on nondenaturing 6% polyacrylamide gels containing 5 μM MgCl2. (D and E) Biotin-labeled 34-mer mismatch substrates were incubated (D) with whole cell extracts (10 μg) or (F) with whole cell extracts (10 μg) and G·T mismatch substrates (lanes 3–5: non-labeled substrates/5′-labeled substrates molar ratio; ×1, ×10, and ×50). Samples were loaded on 10% SDS-page gels, and MSH2 and MSH6 were detected by western blotting.
Mentions: The proofreading function of DNA polymerases plays an important role in correcting replicative mismatch errors. Our results suggest this proofreading occurs at G·5caC pairings but not at other cytosine pairings. Although 5caC forms appropriate base pairs with guanine, we hypothesized that these pairings behave like mismatches (Figure 3A). If this holds true, the mismatch repair (MMR) protein MutS should recognize both pairings as it does G·T mismatches, which are a canonical MutS substrate313233. To test this possibility, we performed electrophoretic mobility shift assays (EMSAs) with Taq MutS and 30-mer DNA substrates containing G·T, G·C, G·5mC, G·5hmC, G·5fC, and G·5caC. We observed a striking difference in MutS binding efficiency between these forms of cytosine. MutS bound G·T and G·5caC pairs (Figure S4A and S4B). The binding preference order was G·T = G·5caC > G·5fC > G·C = G·5mC = G·5hmC. Next, we performed EMSAs with 34-mer G·5caC-containing DNA substrates and human MMR protein MutSα complexes, which consist of MSH2 and MSH6 (Figure S5), because the exonuclease activity of Polδ was observed only on the 5caC templates. The MutSα complex is a human homolog of the MMR protein MutS and is indispensable for the mammalian MMR system3435. MutSα bound to the positive control G·T pairs and to the G·5caC pairs (Figure 3B); addition of excess cold G·T DNA substrates inhibited binding between MutSα and G·5caC DNA substrates (Figure 3C). To confirm this interaction, biotin labeled-G·5caC DNA substrates were incubated with HeLa whole cell extracts and the DNA-bound proteins were pulled down with streptavidin-coated beads; MSH2 and MSH6 were detected by immunoblotting. Results confirmed the MutSα complex recognized G·5caC pairs in DNA substrates (Figures 3D and 3E). Thus, the G·5caC pairs behaved similarly to a G·T mismatch when Polδ synthesized new DNA fragments opposite 5caC, although DNA polymerase correctly incorporated dGTP. In addition, G·5caC pairs may be subjected to MMR in mammalian cells.

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