Methylcytosine and normal cytosine deamination by the foreign DNA restriction enzyme APOBEC3A - PubMed (original) (raw)

. 2012 Oct 5;287(41):34801-8.

doi: 10.1074/jbc.M112.385161. Epub 2012 Aug 15.

Ming Li, Anurag Rathore, Lela Lackey, Emily K Law, Allison M Land, Brandon Leonard, Shivender M D Shandilya, Markus-Frederik Bohn, Celia A Schiffer, William L Brown, Reuben S Harris

Affiliations

Methylcytosine and normal cytosine deamination by the foreign DNA restriction enzyme APOBEC3A

Michael A Carpenter et al. J Biol Chem. 2012.

Abstract

Multiple studies have indicated that the TET oxidases and, more controversially, the activation-induced cytidine deaminase/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC to T activity and 10-fold less C to U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered, whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for nonchromosomal DNA MeC to T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.

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Figures

FIGURE 1.

FIGURE 1.

A3A is a potent single strand DNA deaminase. A, Coomassie gel image of A3A-mycHis and its catalytic mutant in comparison with 0.2–1 μg of BSA. B, assay schematic in which a cytosine in a fluorescently labeled (asterisk) ssDNA substrate is deaminated to uracil and then converted to a shorter reaction product by UDG and NaOH. C, gel image showing the relationship between A3A (2-fold dilutions from 50 n

m

) and ssDNA deaminase activity with 200 n

m

substrate in 60 min. Parallel control reactions had no enzymes, UDG only, or 50 n

m

A3A-E72A. D, reaction series as in C, but dsDNA was used as substrate by preincubating with 1.5-fold excess complement. Parallel control reactions had no enzymes, UDG plus/minus complement, or 50 n

m

A3A with complement. E, quantification of the data in C and D, indicating the amount of product formed as a function of enzyme concentration. MW, molecular weight.

FIGURE 2.

FIGURE 2.

A3A is also a potent MeC to T deaminase. A, representative gels of an activity time course of 3 n

m

A3A on 600 n

m

oligonucleotide with a single cytosine (top gel) or MeC (bottom gel). B, quantification by densitometry of data in A and two independent experiments (means ± S.D. with error smaller than the symbol in most instances). C, representative gels of an activity time course of 100 n

m

A3G against 600 n

m

substrate ssDNA with either CC (top gel) or CMeC (bottom gel). D, quantification by densitometry of data in C and two independent experiments (means ± S.D. with error smaller than the symbol in most instances). Quantification of the −1 C in the sequence is also indicated, demonstrating that this C is not deaminated by A3G (black symbols mostly hidden).

FIGURE 3.

FIGURE 3.

A3A mutants retain C to U and MeC to T activity. A, alignment of the zinc-binding motif from A3A, A3G, and AID. Asterisks and green boxes indicate zinc-binding cysteines. A3G α3 is underlined. B, representative gels showing a time course of 3 n

m

A3AΔWG on 600 n

m

substrate ssDNA with either a single C (top) or MeC (bottom). C, quantification of three independent assays with A3AΔWG against C and MeC (mean ± S.D.). D, alignment A3A N-terminal residues and the corresponding A3G region. The methionines discussed in the text are highlighted. A3G α1 is underlined. E, representative gels indicating the activity of 3 n

m

A3A13–199 on 600 n

m

C oligonucleotide (top) or MeC oligonucleotide (bottom). F, quantification of three independent assays with A3A13–199 against C and MeC ssDNA substrates (mean ± S.D.).

FIGURE 4.

FIGURE 4.

Endogenous A3A deaminates both normal C and MeC foreign DNA substrates. A, representative image of endogenous A3A localization in IFNα-treated THP1 cells. B, genomic MeC levels in IFNα-treated THP1 cells and primary monocytes. The inset shows a representative immunoblot of A3A with tubulin as a loading control. C, agarose gel image of MeC and normal C plasmid DNA substrates treated as shown. HM, CCC, and Cut indicate higher mass, covalently closed circular, and cleaved plasmid species, respectively. D, hypermutation profiles of MeC and normal C plasmid substrates. Each analysis consisted of >18 independent sequences (GFP nucleotides 516–720) and >380 C/G to T/A mutations. Red and green bars indicate mutations within CpG and other cytosine dinucleotide motifs, respectively. Asterisks denote highly mutated 5′-T

C

G sites. E, quantification of A3A mRNA and protein levels in mock or IFNα-treated THP1 cells stably expressing control (shCon) or A3A knockdown (shA3A) short hairpin RNAs. F, ssDNA C (top panel) and MeC (bottom panel) deaminase activity of extracts from cells in E. Parallel negative (Neg) and positive (Pos) control reactions used enzyme buffer minus and plus partially purified A3A, respectively. Oligonucleotide impurity is responsible for the low level of background product. PMA, phorbol 12-myristate 13-acetate.

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