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Effects of tet-induced oxidation products of 5-methylcytosine on DNA replication in mammalian cells.

Ji D, You C, Wang P, Wang Y - Chem. Res. Toxicol. (2014)

Bottom Line: These oxidized derivatives of 5mdC may not only act as intermediates of active cytosine demethylation in mammals but also serve as epigenetic marks on their own.It remains unclear how 5hmdC, 5fdC, and 5cadC affect DNA replication in mammalian cells.The lack of pronounced perturbation of these oxidized 5mdC derivatives on DNA replication is consistent with their roles in epigenetic regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California , Riverside, California 92521, United States.

ABSTRACT
Recently 5-hydroxymethyl-2'-deoxycytidine (5hmdC), 5-formyl-2'-deoxycytidine (5fdC), and 5-carboxyl-2'-deoxycytidine (5cadC) were discovered in mammalian DNA as oxidation products of 5-methyl-2'-deoxycytidine (5mdC) induced by the ten-eleven translocation family of enzymes. These oxidized derivatives of 5mdC may not only act as intermediates of active cytosine demethylation in mammals but also serve as epigenetic marks on their own. It remains unclear how 5hmdC, 5fdC, and 5cadC affect DNA replication in mammalian cells. Here, we examined the effects of the three modified nucleosides on the efficiency and accuracy of DNA replication in HEK293T human kidney epithelial cells. Our results demonstrated that a single, site-specifically incorporated 5fdC or 5cadC conferred modest drops, by approximately 30%, in replication bypass efficiency without inducing detectable mutations in human cells, whereas replicative bypass of 5hmdC is both accurate and efficient. The lack of pronounced perturbation of these oxidized 5mdC derivatives on DNA replication is consistent with their roles in epigenetic regulation of gene expression.

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In-vivo replication studies of 5mdC and its oxidizedderivatives in HEK293T cells. (A) Representative PAGE gel image showingthe restriction fragments of PCR products of the progeny genome emanatingfrom the replication of 5mdC-, 5hmdC-, 5fdC-, and 5cadC-containingplasmids in HEK293T cells treated with human TDG siRNA or controlnontargeting siRNA. (B) The bypass efficiencies of the modified cytosinesin HEK293T cells. The black bars represent the data from cells treatedwith control siRNA, whereas the gray bars designate the data fromcells treated with hTDG siRNA. The data represent the mean and standarddeviation of results from three independent replication experiments.“*”, p < 0.05. The p values were calculated by using two-tailed, unpaired Student’s t test. The horizontal lines above the bar graph indicatethe pairs of data for which the p values were calculated.
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fig3: In-vivo replication studies of 5mdC and its oxidizedderivatives in HEK293T cells. (A) Representative PAGE gel image showingthe restriction fragments of PCR products of the progeny genome emanatingfrom the replication of 5mdC-, 5hmdC-, 5fdC-, and 5cadC-containingplasmids in HEK293T cells treated with human TDG siRNA or controlnontargeting siRNA. (B) The bypass efficiencies of the modified cytosinesin HEK293T cells. The black bars represent the data from cells treatedwith control siRNA, whereas the gray bars designate the data fromcells treated with hTDG siRNA. The data represent the mean and standarddeviation of results from three independent replication experiments.“*”, p < 0.05. The p values were calculated by using two-tailed, unpaired Student’s t test. The horizontal lines above the bar graph indicatethe pairs of data for which the p values were calculated.

Mentions: We employed our previously described shuttle vector method to investigatehow the modified 5mdC derivatives perturb the efficiency and fidelityof DNA replication in human cells.23−25 To this end, we firstconstructed the 5mdC-, 5hmdC-, 5fdC- and 5cadC-containing double-strandedplasmids, as well as the control vector housing a 5mdC at the correspondingsite (Figure 2A). We incorporated a C/C mismatchtwo nucleotides away from the modified nucleoside site (Figure 2A) so as to differentiate the replication productsof the modified cytosine-containing strand from that of the unmodifiedcomplementary strand. The modified nucleoside-bearing plasmids andthe 5mdC-containing control plasmid were transfected individuallyinto HEK293T cells (Figure 2B). After in-vivo replication for 24 h, the progenies of the plasmidswere isolated from human cells, and residual unreplicated plasmidswere removed by a combined treatment with DpnI and exonuclease IIIas described previously.27,28 The progeny genomeswere subsequently amplified using a pair of PCR primers spanning themodified cytosine site. The resulting PCR products were digested withrestriction enzymes (i.e., NcoI and SfaNI) and subjected to PAGE andLC-MS/MS analyses for product identification and quantification (Figure 2B). The bypass efficiencies were calculated fromthe ratio of the restriction products from the modified 5mdC-containingstrand over that of the unmodified complementary strand and normalizedto the corresponding ratio obtained for the 5mdC-containing plasmid(Figure 3A).24


Effects of tet-induced oxidation products of 5-methylcytosine on DNA replication in mammalian cells.

Ji D, You C, Wang P, Wang Y - Chem. Res. Toxicol. (2014)

In-vivo replication studies of 5mdC and its oxidizedderivatives in HEK293T cells. (A) Representative PAGE gel image showingthe restriction fragments of PCR products of the progeny genome emanatingfrom the replication of 5mdC-, 5hmdC-, 5fdC-, and 5cadC-containingplasmids in HEK293T cells treated with human TDG siRNA or controlnontargeting siRNA. (B) The bypass efficiencies of the modified cytosinesin HEK293T cells. The black bars represent the data from cells treatedwith control siRNA, whereas the gray bars designate the data fromcells treated with hTDG siRNA. The data represent the mean and standarddeviation of results from three independent replication experiments.“*”, p < 0.05. The p values were calculated by using two-tailed, unpaired Student’s t test. The horizontal lines above the bar graph indicatethe pairs of data for which the p values were calculated.
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Related In: Results  -  Collection

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fig3: In-vivo replication studies of 5mdC and its oxidizedderivatives in HEK293T cells. (A) Representative PAGE gel image showingthe restriction fragments of PCR products of the progeny genome emanatingfrom the replication of 5mdC-, 5hmdC-, 5fdC-, and 5cadC-containingplasmids in HEK293T cells treated with human TDG siRNA or controlnontargeting siRNA. (B) The bypass efficiencies of the modified cytosinesin HEK293T cells. The black bars represent the data from cells treatedwith control siRNA, whereas the gray bars designate the data fromcells treated with hTDG siRNA. The data represent the mean and standarddeviation of results from three independent replication experiments.“*”, p < 0.05. The p values were calculated by using two-tailed, unpaired Student’s t test. The horizontal lines above the bar graph indicatethe pairs of data for which the p values were calculated.
Mentions: We employed our previously described shuttle vector method to investigatehow the modified 5mdC derivatives perturb the efficiency and fidelityof DNA replication in human cells.23−25 To this end, we firstconstructed the 5mdC-, 5hmdC-, 5fdC- and 5cadC-containing double-strandedplasmids, as well as the control vector housing a 5mdC at the correspondingsite (Figure 2A). We incorporated a C/C mismatchtwo nucleotides away from the modified nucleoside site (Figure 2A) so as to differentiate the replication productsof the modified cytosine-containing strand from that of the unmodifiedcomplementary strand. The modified nucleoside-bearing plasmids andthe 5mdC-containing control plasmid were transfected individuallyinto HEK293T cells (Figure 2B). After in-vivo replication for 24 h, the progenies of the plasmidswere isolated from human cells, and residual unreplicated plasmidswere removed by a combined treatment with DpnI and exonuclease IIIas described previously.27,28 The progeny genomeswere subsequently amplified using a pair of PCR primers spanning themodified cytosine site. The resulting PCR products were digested withrestriction enzymes (i.e., NcoI and SfaNI) and subjected to PAGE andLC-MS/MS analyses for product identification and quantification (Figure 2B). The bypass efficiencies were calculated fromthe ratio of the restriction products from the modified 5mdC-containingstrand over that of the unmodified complementary strand and normalizedto the corresponding ratio obtained for the 5mdC-containing plasmid(Figure 3A).24

Bottom Line: These oxidized derivatives of 5mdC may not only act as intermediates of active cytosine demethylation in mammals but also serve as epigenetic marks on their own.It remains unclear how 5hmdC, 5fdC, and 5cadC affect DNA replication in mammalian cells.The lack of pronounced perturbation of these oxidized 5mdC derivatives on DNA replication is consistent with their roles in epigenetic regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California , Riverside, California 92521, United States.

ABSTRACT
Recently 5-hydroxymethyl-2'-deoxycytidine (5hmdC), 5-formyl-2'-deoxycytidine (5fdC), and 5-carboxyl-2'-deoxycytidine (5cadC) were discovered in mammalian DNA as oxidation products of 5-methyl-2'-deoxycytidine (5mdC) induced by the ten-eleven translocation family of enzymes. These oxidized derivatives of 5mdC may not only act as intermediates of active cytosine demethylation in mammals but also serve as epigenetic marks on their own. It remains unclear how 5hmdC, 5fdC, and 5cadC affect DNA replication in mammalian cells. Here, we examined the effects of the three modified nucleosides on the efficiency and accuracy of DNA replication in HEK293T human kidney epithelial cells. Our results demonstrated that a single, site-specifically incorporated 5fdC or 5cadC conferred modest drops, by approximately 30%, in replication bypass efficiency without inducing detectable mutations in human cells, whereas replicative bypass of 5hmdC is both accurate and efficient. The lack of pronounced perturbation of these oxidized 5mdC derivatives on DNA replication is consistent with their roles in epigenetic regulation of gene expression.

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