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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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MSH2 knockdown rescued the cell death response in TDG-knockdown cells.(A) Western blotting of lysates prepared from siRNA-transfected HeLa cells using antibodies specific for the indicated proteins. Actin served as a loading control. (B) Mass spectrometric quantification of 5caC in siRNA-transfected HeLa cells. Data represent the average of at least three independent experiments and statistically analyzed by Student's t test (*p < 0.05). Error bars represent standard error of the mean. Three days after siRNA transfection, the percentage of apoptotic cells (C) and the number of viable cells (D) were calculated. Experiments were repeated six times and statistically analyzed by Student's t test (N = 6, *p < 0.01). Error bars represent standard deviation (SD). (E and F) 293 cells expressing mouse Tet1 under the control of Doxycycline were transfected with siRNA in the absence or presence of Doxycycline. Three days after siRNA transfection, expression of indicated proteins was examined by specific antibodies (E) and the number of viable cells was calculated (F). Experiments were repeated three times. Error bars represent SD. (G) Model for DNA damage effects of 5caC in DNA replication via abortive mismatch repair.
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f4: MSH2 knockdown rescued the cell death response in TDG-knockdown cells.(A) Western blotting of lysates prepared from siRNA-transfected HeLa cells using antibodies specific for the indicated proteins. Actin served as a loading control. (B) Mass spectrometric quantification of 5caC in siRNA-transfected HeLa cells. Data represent the average of at least three independent experiments and statistically analyzed by Student's t test (*p < 0.05). Error bars represent standard error of the mean. Three days after siRNA transfection, the percentage of apoptotic cells (C) and the number of viable cells (D) were calculated. Experiments were repeated six times and statistically analyzed by Student's t test (N = 6, *p < 0.01). Error bars represent standard deviation (SD). (E and F) 293 cells expressing mouse Tet1 under the control of Doxycycline were transfected with siRNA in the absence or presence of Doxycycline. Three days after siRNA transfection, expression of indicated proteins was examined by specific antibodies (E) and the number of viable cells was calculated (F). Experiments were repeated three times. Error bars represent SD. (G) Model for DNA damage effects of 5caC in DNA replication via abortive mismatch repair.

Mentions: In the MMR system, exonuclease I removed the daughter DNA strand but could not remove template DNA containing the modified cytosine. Thus, the “offending” site persisted in the template. The ensuing abortive turnover of new DNA may result in a death response. Earlier studies have shown that TDG binds to these G·5caC pairs36 and excises the modified cytosine20. To investigate the effects of these G·T mismatch-mimicking base pairs in mammalian cells in vivo, we confirmed the expression levels of Tets, TDG, and MSH2 in various human cells (Figure S6) and then knocked down TDG expression, thereby inducing the accumulation of G·5caC base pairs and observed the viable cells in TDG-knockdown cultures (Figure 4A and Figure S7). As expected, 5caC was induced in TDG-knockdown cells (Figure 4B and Figure S8) versus control-knockdown or MSH2-knockdown cells. TDG-knockdown HeLa cells exhibited elevated apoptotic cell population and decreased number of surviving cells (Figure 4C and 4D), indicating the accumulated G·5caC base pairs are recognized by MMR, which induces the effects of DNA damage. This phenotype was partially rescued by knockdown of MSH2 expression. Once again, because Tet1-overexpressed 293 cells exhibit increased 5hmC and 5caC levels19, we investigated the effects of TDG knockdown in Tet1-overexpressed 293 cells (Figure 4E and Figure S9). When Tet1 expression was induced by treatment with doxycycline (Dox), cell number was reduced in all cases, indicating that Tet1-modifying cytosines behave as DNA lesions (Figure 4F). TDG knockdown leads to reduced cell numbers that were rescued by TDG-MSH2 double knockdown (Figure 4F). Thus, MMR was required for these DNA damage effects, which result in cell proliferation defects and decreased cell number.


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)

MSH2 knockdown rescued the cell death response in TDG-knockdown cells.(A) Western blotting of lysates prepared from siRNA-transfected HeLa cells using antibodies specific for the indicated proteins. Actin served as a loading control. (B) Mass spectrometric quantification of 5caC in siRNA-transfected HeLa cells. Data represent the average of at least three independent experiments and statistically analyzed by Student's t test (*p < 0.05). Error bars represent standard error of the mean. Three days after siRNA transfection, the percentage of apoptotic cells (C) and the number of viable cells (D) were calculated. Experiments were repeated six times and statistically analyzed by Student's t test (N = 6, *p < 0.01). Error bars represent standard deviation (SD). (E and F) 293 cells expressing mouse Tet1 under the control of Doxycycline were transfected with siRNA in the absence or presence of Doxycycline. Three days after siRNA transfection, expression of indicated proteins was examined by specific antibodies (E) and the number of viable cells was calculated (F). Experiments were repeated three times. Error bars represent SD. (G) Model for DNA damage effects of 5caC in DNA replication via abortive mismatch repair.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: MSH2 knockdown rescued the cell death response in TDG-knockdown cells.(A) Western blotting of lysates prepared from siRNA-transfected HeLa cells using antibodies specific for the indicated proteins. Actin served as a loading control. (B) Mass spectrometric quantification of 5caC in siRNA-transfected HeLa cells. Data represent the average of at least three independent experiments and statistically analyzed by Student's t test (*p < 0.05). Error bars represent standard error of the mean. Three days after siRNA transfection, the percentage of apoptotic cells (C) and the number of viable cells (D) were calculated. Experiments were repeated six times and statistically analyzed by Student's t test (N = 6, *p < 0.01). Error bars represent standard deviation (SD). (E and F) 293 cells expressing mouse Tet1 under the control of Doxycycline were transfected with siRNA in the absence or presence of Doxycycline. Three days after siRNA transfection, expression of indicated proteins was examined by specific antibodies (E) and the number of viable cells was calculated (F). Experiments were repeated three times. Error bars represent SD. (G) Model for DNA damage effects of 5caC in DNA replication via abortive mismatch repair.
Mentions: In the MMR system, exonuclease I removed the daughter DNA strand but could not remove template DNA containing the modified cytosine. Thus, the “offending” site persisted in the template. The ensuing abortive turnover of new DNA may result in a death response. Earlier studies have shown that TDG binds to these G·5caC pairs36 and excises the modified cytosine20. To investigate the effects of these G·T mismatch-mimicking base pairs in mammalian cells in vivo, we confirmed the expression levels of Tets, TDG, and MSH2 in various human cells (Figure S6) and then knocked down TDG expression, thereby inducing the accumulation of G·5caC base pairs and observed the viable cells in TDG-knockdown cultures (Figure 4A and Figure S7). As expected, 5caC was induced in TDG-knockdown cells (Figure 4B and Figure S8) versus control-knockdown or MSH2-knockdown cells. TDG-knockdown HeLa cells exhibited elevated apoptotic cell population and decreased number of surviving cells (Figure 4C and 4D), indicating the accumulated G·5caC base pairs are recognized by MMR, which induces the effects of DNA damage. This phenotype was partially rescued by knockdown of MSH2 expression. Once again, because Tet1-overexpressed 293 cells exhibit increased 5hmC and 5caC levels19, we investigated the effects of TDG knockdown in Tet1-overexpressed 293 cells (Figure 4E and Figure S9). When Tet1 expression was induced by treatment with doxycycline (Dox), cell number was reduced in all cases, indicating that Tet1-modifying cytosines behave as DNA lesions (Figure 4F). TDG knockdown leads to reduced cell numbers that were rescued by TDG-MSH2 double knockdown (Figure 4F). Thus, MMR was required for these DNA damage effects, which result in cell proliferation defects and decreased cell number.

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