Efficient deamination of 5-methylcytosines in DNA by human APOBEC3A, but not by AID or APOBEC3G - PubMed (original) (raw)
Efficient deamination of 5-methylcytosines in DNA by human APOBEC3A, but not by AID or APOBEC3G
Priyanga Wijesinghe et al. Nucleic Acids Res. 2012 Oct.
Abstract
The AID/APOBEC family of enzymes in higher vertebrates converts cytosines in DNA or RNA to uracil. They play a role in antibody maturation and innate immunity against viruses, and have also been implicated in the demethylation of DNA during early embryogenesis. This is based in part on reported ability of activation-induced deaminase (AID) to deaminate 5-methylcytosines (5mC) to thymine. We have reexamined this possibility for AID and two members of human APOBEC3 family using a novel genetic system in Escherichia coli. Our results show that while all three genes show strong ability to convert C to U, only APOBEC3A is an efficient deaminator of 5mC. To confirm this, APOBEC3A was purified partially and used in an in vitro deamination assay. We found that APOBEC3A can deaminate 5mC efficiently and this activity is comparable to its C to U deamination activity. When the DNA-binding segment of AID was replaced with the corresponding segment from APOBEC3A, the resulting hybrid had much higher ability to convert 5mC to T in the genetic assay. These and other results suggest that the human AID deaminates 5mC's only weakly because the 5-methyl group fits poorly in its DNA-binding pocket.
Figures
Figure 1.
(A) Sequence context of 5mC in kan alleles. The five different methylation contexts created in the kan gene through changes in base sequence or methylation are shown. Three different kan alleles are present in E. coli strains BH300, BH400 and BH500. The gene for the MTase M.HpaII, M.MspI or Dcm was introduced in these strains to methylate one of the cytosines in a proline codon (underlined in the figure). The methylated cytosine is indicated by ‘me’ above the C. The five bases unique to each sequence context are indicated by a bracket below the sequence. 5mC to thymine mutation changes the proline codon to either leucine or serine and changes the cellular phenotype from kanamycin-sensitive (KanS) to kanamycin-resistant (KanR). (B) Effects of MTases on KanR revertant frequency. The frequency of revertants with or without the presence of a MTase is shown. The bacterial host used (BH300, BH400 or BH500) and the relevant methylated sequences are shown. The genes for M.HpaII, M.MspI or Dcm were introduced in the cells to methylate the DNA. ‘M’ is 5-mC and the horizontal line within the data points is the mean value.
Figure 2.
(A) Very short-patch repair of T•G mispairs in E. coli. The pathway (33) by which E. coli VSP repair replaces T•G mispairs created by the deamination of 5mCs in the Dcm sequence context with C•G is shown. Deamination of one of the 5mCs in a Dcm sequence context creates a T•G mispair. The Vsr endonuclease hydrolyzes the phosphodiester linkage 5′ of the mispaired T and DNA polymerase I (PolI) and DNA ligase complete the reaction. (B) KanR revertant frequency with and without VSP repair. The revertant frequencies in the presence of Dcm alone or with both Dcm and Vsr in the cells (BH400) are shown and are compared with the frequency in the absence of either enzyme. The horizontal line within the data points is the median value.
Figure 3.
(A) Cytosine deamination by AID. KanR revertant frequencies in BH500 cells expressing AID alone or AID and UGI. The horizontal line within the data points is the median value. (B) 5mC deamination by AID. KanR revertant frequencies in BH500 cells expressing AID, E58A mutant of AID or empty vector are shown. The horizontal line within the data points is the median value. (C) Quantification of genomic uracils. The amount of genomic uracil created by AID alone or AID and UGI is shown. The error bars indicate the standard deviation.
Figure 4.
(A) Comparison of cytosine deamination by AID and A3G. KanR revertant frequencies in BH300 cells expressing AID, E58A mutant of AID or A3G are shown. The horizontal line within the data points is the median value. (B) Comparison of 5mC deamination by AID and A3G. KanR revertant frequencies in BH300 cells expressing vector, AID or A3G along with M.HpaII are shown. The horizontal line within the data points is the median value.
Figure 5.
(A) Comparison of cytosine deamination by AID and A3A. KanR revertant frequencies in BH500 cells expressing AID, E58A mutant of AID or A3A are shown. The horizontal line within the data points is the median value. (B) Comparison of 5mC deamination by AID and A3A. KanR revertant frequencies in BH500 cells expressing AID, E58A mutant of AID or A3A along with M.HpaII are shown. (C) 5mC deamination by A3A. KanR revertant frequencies in BH500 cells expressing A3A, E72A mutant of A3A or empty vector are shown. The horizontal line within the data points is the median value.
Figure 6.
(A) Cytosine and 5mC deamination by A3A. Fluorescently labeled DNA oligomers with a single C or 5mC were treated with A3A or A3A-E72A. The C-containing oligomer was treated with UDG and cleaved while the 5mC-containing oligomer was hybridized to its complement, treated with TDG and cleaved. (B) Kinetics of cytosine and 5mC deamination by A3A. Fluorescently labeled DNA oligomers with a single C or 5mC were treated with A3A and the reactions were stopped at indicated times. The complementary strand was hybridized to the oligomer and UDG or TDG was added to cleave the DNA strand. DNA duplex containing U•G or T•G mispairs respectively serve as controls for the efficiency of reactions with UDG and TDG. (C) Quantification of data in part B above. The data are normalized for the efficiency of UDG and TDG reactions, and the signal at zero time was subtracted from all data points.
Figure 7.
(A) Domain swap between AID and A3A. The sequence of the putative DBDs of AID (21,40,41) and A3A are shown schematically. The DBD of A3A was identified by aligning the sequence of this protein with the sequence of AID and of the carboxy-terminal domain of A3G. AID-A3AR2 contains all of AID except its DBD which is replaced with eight amino acid DBD from A3A. The numbers above and below the sequences are amino acid residue numbers. (B) Comparison of 5mC deamination by AID, A3A and AID-A3AR2. KanR revertant frequencies in BH500 cells expressing the different proteins are shown. The horizontal line within the data points is the median value.
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