The cytidine deaminase under-representation reporter (CDUR) as a tool to study evolution of sequences under deaminase mutational pressure

Abstract

Background: Activation induced deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) are deaminases that mutate C to U on single-stranded DNA (ssDNA). AID is expressed primarily in germinal center B-cells, where it facilitates affinity maturation and class-switch recombination. APOBEC3 are a family of anti-viral proteins that act as part of the intrinsic immune response. In both cases, there are particular sequence motifs, also known as "mutation motifs", to which these deaminases prefer to bind and mutate.

Results: We present a program, the cytidine deaminase under-representation reporter (CDUR) designed to statistically determine whether a given sequence has an under/over-representation of these mutation motifs. CDUR shows consitency with other studies of mutation motifs, as we show by analyzing sequences from the adeno-associated virus 2 (AAV2) and human papillomavirus (HPV).

Conclusion: Using various shuffling mechanisms to generate different null model distributions, we can tailor CDUR to correct for metrics such as GC-content, dinucleotide frequency, and codon bias.

Keywords: AID; APOBEC; Deaminase; Mutation motifs; Virus.

Fig. 1

gc3 shuffle method. The choice of codons in the 4th nucleotide in the sequence (Ile) was determined by the probabilities as follows: since there is an overall GC content of 60% at the 3rd position of the codons in the subject sequence, the ATC codon will be chosen with 0.6 probability. Since the AT content is then 0.4, the other two codons ATT and ATA are chosen randomly with equal probability, conditional on the 40% AT content. Note that the shuffling occurs iteratively throughout sequence, not just one codon at a time

Fig. 2

n3 shuffle method. Third codon positions are first categorized into whether they belong to Y, R, H, or N nucleotides, then assigned to the corresponding set (Type vs Set table). Then nucleotides within each set are shuffled (Type vs Shuffle table) to produce the shuffled sequence. Certain nucleotides can belong to two sets (here two nucleotides belong to both H and N) and are shuffled twice

Fig. 3

dn23 shuffle method. First the dinucleotide frequency is calculated for the 2nd and 3rd codon positions of the original sequence. Then for each amino acid, codons are chosen based on the appropriately normalized probabilities for the dinucleotides available for that amino acid

Fig. 4

Rep-68 Under-representation. Histograms showing the results of CDUR applied to the AAV2 Rep-68 gene using the gc3 shuffle method. In each graph, the filled black bars represent the “below” portion, that is, the number of shuffled sequences with that given metric below that of the one computed for the input. The red line shows the Normal distribution approximation given the mean and standard deviation (SD) from that calculation. a The mutation motif counting graph (belowTAC). The observed number of TAC mutation motifs is 20 (which includes the GTA mutation motifs on the bottom strand). The black bars are all shuffled sequences with fewer observed mutation motifs than our input. b The replacement transition graph (RepTr_belowTAC), also shown with the Normal approximation. c The replacement transition fraction graph (RepTrFrac_belowTAC). Our sequence had an observed RepTrFrac of 0.25, so the sequences with a lower RepTrFrac are filled in with black (see Table 2 for complete results)

Fig. 5

E6 Under-representation. Histograms showing the results of CDUR applied to the HPV E6 gene using the gc3 shuffle method. All subplots (a-c) and annotations are equivalent to those of Fig. 4, but for this gene. In contrast to Fig. 4 however, rather than under-representation, we see a neutral representation for TAC mutation motifs. (See Table 3 for complete results)