Evolution of the primate APOBEC3A cytidine deaminase gene and identification of related coding regions

Abstract

The APOBEC3 gene cluster encodes six cytidine deaminases (A3A-C, A3DE, A3F-H) with single stranded DNA (ssDNA) substrate specificity. For the moment A3A is the only enzyme that can initiate catabolism of both mitochondrial and nuclear DNA. Human A3A expression is initiated from two different methionine codons M1 or M13, both of which are in adequate but sub-optimal Kozak environments. In the present study, we have analyzed the genetic diversity among A3A genes across a wide range of 12 primates including New World monkeys, Old World monkeys and Hominids. Sequence variation was observed in exons 1-4 in all primates with up to 31% overall amino acid variation. Importantly for 3 hominids codon M1 was mutated to a threonine codon or valine codon, while for 5/12 primates strong Kozak M1 or M13 codons were found. Positive selection was apparent along a few branches which differed compared to positive selection in the carboxy-terminal of A3G that clusters with A3A among human cytidine deaminases. In the course of analyses, two novel non-functional A3A-related fragments were identified on chromosome 4 and 8 kb upstream of the A3 locus. This qualitative and quantitative variation among primate A3A genes suggest that subtle differences in function might ensue as more light is shed on this increasingly important enzyme.

Figure 1. Alignment of primate APOBEC3A proteins.

Twelve primate sequences were compared to Homo sapiens used as reference. Only differences are shown. Hyphens denote gaps introduced to maximize sequence identity. The numbering corresponds to that of the human sequence. The letters a, b, c are added to adjacent residue to accommodate insertions. Red denotes the first (M1, exon 1) and second initiation start codons (M13, exon 2). The crucial cytidine deaminase motif residues are highlighted in magenta. Positively and negatively selected codon sites are in blue and green respectively. The predicted secondary structure motifs for hA3A are underlined.

Figure 2. Phylogeny of primate A3A and A3Gc cytidine deaminases.

A) Neighbor-joining tree based on A3A primate sequences presented in Figure 1. Only bootstrap values >70 are indicated. The nomenclature for the Kozak initiation sites (A: adequate, N: null, S: strong) are from Table 1. B) Neighbor-joining tree based on A3Gc primate sequences taken in the literature. For both, red branches correspond to class d_N/d_S>1 (p>0.9) while the remainder correspond to classes d_N/d_S<1. C) The accepted divergence of the Great Apes.

Figure 3. No cytidine deaminase activity associated with pΨA3chr4.

A) Alignment of exon 3 of ΨA3chr4 proteins to human A3Bc, A3Gc and A3A. Differences with respect to ΨA3chr4 are highlighted in red. B) Neighbor-joining tree based on the exon 3 of human A3 genes. Only bootstrap values of >70 are given. C) Immunostaining of the V5-tagged pΨA3chr4 and A3A proteins with DAPI nuclear counterstain. D) Agarose gel of 3DPCR products across a denaturation temperature gradient from 93 to 85°C of X region of HBV. pCayw is an infectious molecular clone along with the empty expression vector, along with pΨA3chr4. M; molecular weight markers. The white asterisks denote the last amplification product obtained at 91.8°C. E) Agarose gel of 3DPCR products across a denaturation temperature gradient from 89 to 79°C on MT-COI gene. The white asterisks denote the last amplification product of MT-COI obtained at 87°C.