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J. Biol. Chem., Vol. 283, Issue 25, 17428-17439, June 20, 2008

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Impact of Phosphorylation and Phosphorylation-null Mutants on the Activity and Deamination Specificity of Activation-induced Cytidine Deaminase^*

Phuong Pham, Marcus B. Smolka, Peter Calabrese, Alice Landolph, Ke Zhang^¶, Huilin Zhou, and Myron F. Goodman¹

From the Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, California 90089-2910, the Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093, and the ^¶Division of Clinical Immunology/Allergy, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90095

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination in B cells by deaminating C U on transcribed DNA. Here we analyze the role of phosphorylation and phosphorylation-null mutants on the biochemical behavior of AID, including enzyme specific activity, processivity, deamination spectra, deamination motif specificity, and transcription-dependent deamination in the presence and absence of RPA. We show that a small fraction of recombinant human AID expressed in Sf9 insect cells is phosphorylated at previously identified residues Ser³⁸ and Thr²⁷ and also at Ser⁴¹ and Ser⁴³. S43P AID has been identified in a patient with hyper-IgM immunodeficiency syndrome. Ser-substituted phosphorylation-null mutants (S38A, S41A, S43A, and S43P) exhibit wild type (WT) activity on single-stranded DNA. Deamination of transcribed double-stranded DNA is similar for WT and mutant AID and occurs with or without RPA. Although WT and AID mutants catalyze processive deamination favoring canonical WRC hot spot motifs (where W represents A/T and R is A/G), their deamination spectra differ significantly. The differences between the WT and AID mutants appear to be caused by the replacement of Ser as opposed to an absence of phosphorylation. The spectral differences reflect a marked change in deamination efficiencies in two motifs, GGC and AGC, which are preferred by mutant AID but disfavored by WT AID. Both motifs occur with exceptionally high frequency in human switch regions, suggesting a possible relationship between AID deamination specificity and a loss of antibody diversification.

Received for publication, March 17, 2008

^* This work was supported, in whole or in part, by National Institutes of Health Grants ES013192 and R37GM21422 (to M. F. G.) and Grant GM080469. This work was also supported by the Ludwig Institute for Cancer Research (to H. Z. and M. B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed: Depts. of Biological Sciences and Chemistry, RRI-201, University of Southern California, Los Angeles, CA 90089-2910. Tel.: 213-740-5190; Fax: 213-821-1138; E-mail: mgoodman{at}usc.edu.

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