Mutations in human AID differentially affect its ability to deaminate cytidine and 5-methylcytidine in ssDNA substrates in vitro

Lucyna Budzko,Paulina Jackowiak,Marek Figlerowicz,Janusz M Bujnicki,Karol Kamel,Joanna Sarzynska

doi:10.1038/s41598-017-03936-x

Abstract

Activation-induced cytidine deaminase (AID) is known for its established role in antibody production. AID induces the diversification of antibodies by deaminating deoxycytidine (C) within immunoglobulin genes. The capacity of AID to deaminate 5-methyldeoxycytidine (5 mC) and/or 5-hydroxymethyldeoxycytidine (5 hmC), and consequently AID involvement in active DNA demethylation, is not fully resolved. For instance, structural determinants of AID activity on different substrates remain to be identified. To better understand the latter issue, we tested how mutations in human AID (hAID) influence its ability to deaminate C, 5 mC, and 5 hmC in vitro. We showed that each of the selected mutations differentially affects hAID’s ability to deaminate C and 5 mC. At the same time, we did not observe hAID activity on 5 hmC. Surprisingly, we found that the N51A hAID mutant, with no detectable activity on C, efficiently deaminated 5 mC, which may suggest different requirements for C and 5 mC deamination. Homology modeling and molecular dynamics simulations revealed that the pattern of enzyme-substrate recognition is one of the important factors determining enzyme activity on C and 5 mC. Consequently, we have proposed mechanisms that explain why wild type hAID more efficiently deaminates C than 5 mC in vitro and why 5 hmC is not deaminated.

Highlights

Activation-induced cytidine deaminase (AID) acts exclusively on ssDNA and deaminates C preferentially within WRCY or WRC motifs (W = A/T; R = A/G; Y = C/T)[6,7,8,9]
There are three possible scenarios of 5 mC modification and exchange into C21, 26: (i) direct deamination of 5 mC to T by AID/APOBEC deaminase(s), followed by processing of the T-G mismatch by base excision repair (BER) machinery[27, 28]; (ii) oxidation of 5 mC to 5 hmC catalyzed by TET proteins, followed by deamination of 5 hmC to 5-hydroxymethyldeoxyuracil (5 hmU) by AID/ APOBEC deaminase(s) and 5 hmU removal by BER28, 29; and (iii) oxidation of 5 mC to 5 hmC catalyzed by TET proteins, followed by iterative oxidation that leads to the formation of 5-formyldeoxycytidine (5 fC) and 5-carboxyldeoxycytidine (5caC)
It was previously reported that, in vitro, the R50A mutant exhibited decreased deaminase activity on C17, whereas N51A failed to show this activity[17, 18], and R190X was more active than wt hAID15

Summary

Introduction

The mean lifetime for AID bound to ssDNA is ∼5 min[14] Both SHM and CSR depend on AID deamination activity, the amino acid mutations that alter the latter may differentially affect these processes (Supplementary Table S1). Several mechanisms of active genome demethylation have been proposed[21, 22] According to these mechanisms 5 mC undergoes enzymatic conversion to another modified nucleotide that is subsequently processed by DNA repair machinery. The deamination activity on 5 hmC in vitro has not been confirmed[37, 39]

Methods

Results

Conclusion