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. 2009 Aug 21;284(34):22898-904.
doi: 10.1074/jbc.M109.025536. Epub 2009 Jun 26.

A portable hot spot recognition loop transfers sequence preferences from APOBEC family members to activation-induced cytidine deaminase

Rahul M Kohli  1 , Shaun R Abrams, Kiran S Gajula, Robert W Maul, Patricia J Gearhart, James T Stivers
Affiliations

"VSports最新版本" A portable hot spot recognition loop transfers sequence preferences from APOBEC family members to activation-induced cytidine deaminase

"VSports" Rahul M Kohli et al. J Biol Chem. .

Abstract

Enzymes of the AID/APOBEC family, characterized by the targeted deamination of cytosine to generate uracil within DNA, mediate numerous critical immune responses. One family member, activation-induced cytidine deaminase (AID), selectively introduces uracil into antibody variable and switch regions, promoting antibody diversity through somatic hypermutation or class switching. Other family members, including APOBEC3F and APOBEC3G, play an important role in retroviral defense by acting on viral reverse transcripts. These enzymes are distinguished from one another by targeting cytosine within different DNA sequence contexts; however, the reason for these differences is not known. Here, we report the identification of a recognition loop of 9-11 amino acids that contributes significantly to the distinct sequence motifs of individual family members. When this recognition loop is grafted from the donor APOBEC3F or 3G proteins into the acceptor scaffold of AID, the mutational signature of AID changes toward that of the donor proteins. These loop-graft mutants of AID provide useful tools for dissecting the biological impact of DNA sequence preferences upon generation of antibody diversity, and the results have implications for the evolution and specialization of the AID/APOBEC family VSports手机版. .

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Figures

FIGURE 1.
FIGURE 1.
A protein loop in the AID/APOBEC family is poised for potential interactions with target DNA. A, a role for the hot spot recognition loop. Unliganded APOBEC3G (Protein Data Bank code 3E1U) and the RNA-bound TadA (Protein Data Bank code 2B3J) structures were aligned by the Dali server (43). The structure shown is APOBEC3G with the aligned TadA RNA substrate. The structure suggests a role for a loop (red) in recognition of the nucleotides (−1 position in green) upstream of the deamination target (*). B, alignment of AID/APOBEC family members. An area downstream from the catalytic residues is shown in partial sequence alignment. The structural loop noted in A is poorly conserved (red box) and flanked by highly conserved sequences. AID and the C-terminal catalytic domains of APOBEC3F and -3G are shown schematically with their distinct sequence preferences noted. The active site residues are upstream from this loop and AID residue numbering is noted. The loop variants were constructed by replacing the highlighted loop from AID with that from APOBEC3F (AID-3FL) or APOBEC3G (AID-3GL) and their sequence preferences investigated in this work.
FIGURE 2.
FIGURE 2.
AID-WT targets deamination to WRC hot spot sequences. A, expression of soluble AID-WT. 1-ml cultures from full-length MBP-AID, the C-terminal truncation (MBP-AID-ΔC or AID-WT), and co-expression with the chaperone trigger factor (TF) were separated into soluble (S) and insoluble (IS) fractions demonstrating progressively increasing soluble expression. The elution from the amylose affinity resin is shown (E). B, oligonucleotide deamination assay. An array of substrates (S60-XXC) with a single substrate cytosine in a 60-mer single-strand DNA are incubated with AID-WT and UNG. The substrates include variations where the −1 (X−1) or −2 positions (X−2) are A, G, or T or mC. The resulting abasic sites (^) are cleaved with the endonuclease APE, giving P24 and P36 products. C, AID-WT prefers WRC hot spot sequences. 1 μm substrate was incubated with 1 μg of AID-WT (or no enzyme control) for 3 h. The P24/P36 products generated by UNG and APE treatments were detected on a denaturing gel by staining with SybrGold. S60-AGU serves as a product control for the activity of UNG and APE and as a size standard.
FIGURE 3.
FIGURE 3.
Grafting of a loop from APOBEC enzymes onto AID results in predictable shifts in sequence preference profiles. A, product formation with AID-WT correlates with in vivo sequence preferences. B, loop grafting in AID-3FL, and C, AID-3GL alter sequence preferences. For each condition, 1 μm S60-XXC substrate was subjected to 1 μg of enzyme for 3 h under assay conditions. The products from 3 to 5 replicates of each condition were quantified and the average values for % product formation are shown for AID-WT, AID-3FL, and AID-3GL. The best substrates for each construct are shown in bold italics. The detection limit of the assay is ≤1%. For substrates with 2–5% conversion to product, standard deviation was 1–2%; for 6–10% conversion, standard deviation was 2–3%; for 11–20% conversion, standard deviation was 5–10%; for 21–33% conversion, standard deviation was 6–11%. The data were used to construct the sequence preference profile for AID-WT and the loop graft variants. The in vivo deamination sequence preferences are shown for comparison. These values are derived from previously reported sequences of variable regions of B-cells mutated in ung−/−, msh2−/− mice for AID (12, 24) or from the reported sequencing of hypermutated retroviral genomes for APOBEC3F and APOBEC3G (20).
FIGURE 4.
FIGURE 4.
Loop graft variants alter the mutational targeting conferring resistance to rifampin in E. coli. A, induction of a mutator phenotype in E. coli. Expression of AID or loop variants along with the uracil DNA glycosylase inhibitor leads to an increase in rifampin resistance. The mutagenesis frequency was calculated based on the number of rifampin-resistant colonies per total cells. Shown are the data for eight individual experiments (+) for each expression plasmid. The average mutagenesis frequency is shown with the gray bar graph. The ratio of mutagenesis frequency with AID-WT, AID-3FL, or AID-3GL over the control pET41 plasmid are listed below the plot. B, loop variants have an altered mutational spectrum. The rpoB gene from individual colonies was sequenced to determine the mutation spectrum generated by each enzyme. The total number of sequences obtained were: AID-WT (43), AID-3FL (43), and AID-3GL (36). The mutated position of the rpoB gene is shown, along with the surrounding trinucleotide sequence targeted for deamination with the deaminated cytosine underlined. The plot reports on the frequency of mutation at all detected positions demonstrating the alteration in mutational targeting by AID-3FL and AID-3GL.
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