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

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.

Figure 1

Chemical structures of modified 2′-deoxycytidine derivatives found in mammalian DNA. “dR” represents 2-deoxyribose.

Figure 2

Experimental procedures for the construction of modified cytosine-containing duplex vectors (A) and for determining the effects of the modified cytosine derivatives on DNA replication in cells (B). “X” represents 5mdC, 5hmdC, 5fdC, or 5cadC, and the C:C mismatch site is underlined. “p*” and “p” designate ³²P-labeled and unlabeled phosphate, respectively. The restriction recognition sites are highlighted in bold, and cleavage sites are indicated by arrows.

Figure 3

In-vivo replication studies of 5mdC and its oxidized derivatives in HEK293T cells. (A) Representative PAGE gel image showing the restriction fragments of PCR products of the progeny genome emanating from the replication of 5mdC-, 5hmdC-, 5fdC-, and 5cadC-containing plasmids in HEK293T cells treated with human TDG siRNA or control nontargeting siRNA. (B) The bypass efficiencies of the modified cytosines in HEK293T cells. The black bars represent the data from cells treated with control siRNA, whereas the gray bars designate the data from cells treated with hTDG siRNA. The data represent the mean and standard deviation 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 indicate the pairs of data for which the p values were calculated.