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, 44 (8), 3713-27

An Interplay of the Base Excision Repair and Mismatch Repair Pathways in Active DNA Demethylation

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An Interplay of the Base Excision Repair and Mismatch Repair Pathways in Active DNA Demethylation

Inga Grin et al. Nucleic Acids Res.

Abstract

Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM.

Figures

Figure 1.
Figure 1.
Processing of the oxidation/deamination products of deoxycytosine and 5mC by BER and ncMMR pathways in cell extracts from MEF cells. (A) In vitro reconstitution of repair of plasmids containing stand-alone dU•G, 5caC•G, G•T or 5hmC•G sites. (B) In vitro reconstitution of repair of plasmids containing 5hmU•G mispairs. (C) Comparison of demethylation efficiency at the hemimethylated SmaI reporter site in the experiments presented in panels A and B. The error bars represent the standard deviation (SD) (n ≥ 3).
Figure 2.
Figure 2.
BER activities toward oligonucleotide substrates containing a single modified base in MEF extracts. (A) 5hmU DNA glycosylase and AP endonuclease activities on double- and single-stranded 30 mer RT oligonucleotide substrates containing 5hmU or tetrahydrofuranyl (THF) residues. (B) 5caC DNA glycosylase activity on covalently closed DSO substrates containing 5caC•G base pair. The error bars represent SD (n = 3). For details, see ‘Materials and Methods’ section and Supplementary Figure S3.
Figure 3.
Figure 3.
Demethylation of the pMM2 plasmid substrates containing multiple 5hmU•G mispairs in MEF extracts. The error bars represent SD (n = 3).
Figure 4.
Figure 4.
Reconstitution of the BER and MMR pathways on pMM2 plasmid substrates containing oxidation/deamination products of deoxycytosine and 5mC in cell extracts from BL2 and MEF cells. (A) Efficiency of repair of dU•G, 5caC•G, G•T and 5hmC•G plasmid substrates in BL2 extracts. (B) Efficiency of repair of plasmids containing 5hmU•G mispairs in BL2 extracts. (C) Efficiency of demethylation at the SmaI site of Mult-hmU5[6] and Mult-dU5[6] plasmids in BL2 and MEF cell extracts.
Figure 5.
Figure 5.
Long-patch BER at a single dU•G in cell-free extracts of MEF and BL2 cells. (A) Long-patch BER dependent on 5mC removal in covalently closed circular (ссс) plasmid substrates containing a 5′ adjacent dU residue. (B) Effects of a uracil-DNA glycosylase inhibitor (UGI) on total repair of a single dU•G in MEF extracts.
Figure 6.
Figure 6.
MMR-dependent demethylation of DNA plasmids containing multiple 5mC residues and mispairs in MEF extracts. (A) Schematic representation of the pCMVIG luciferase reporter plasmid containing custom-made lesions and/or methylated CpG in the promoter region. (B) MMR-dependent demethylation of the hemimethylated pCMVIG plasmid containing multiple dU•G and T•G mispairs. For details, see Materials and Methods’ section and Supplementary Material.
Figure 7.
Figure 7.
Expression of the reporter pCMVIG plasmids with a hemimethylated promoter containing multiple dU•G and 5hmU•G mispairs after transfection in MEFs. (A) Relative changes in the reporter expression of pCMVIG-derived plasmids (LpCMVIG-xxx) that was normalized to the expression level of unmodified pCMVIG (bars represent LpCMVIG-xxx/LpCMVIG ratio). (B) Relative reporter expression of hemimethylated plasmids containing dU•G and 5hmU•G mispairs (LpCMVIG-4X-361-hM); the data are normalized to the level of the reporter expression in the presence of the corresponding mispairs in unmethylated plasmids and to the expression levels of hemimethylated pCMVIG-hM and unmodified pCMVIG plasmids (bars represent LpCMVIG-4X-361-hM/LpCMVIG-4X-361/LpCMVIG-hM/LpCMVIG ratio). Original data of the pCMVIG-derived plasmids expressions shown in Supplementary Figure S7.
Figure 8.
Figure 8.
Schematic representation of the interplay between BER and MMR during long-patch DDM. X: deaminated cytosines, their derivatives and other mispairs; M: mutations.

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