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• W2023254248 abstract "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. 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. AID, a B-cell specific protein, is required for tightly regulated mechanisms of Ig antibody diversification, somatic hypermutation, class switch recombination, and gene conversion. SHM 2The abbreviations used are: SHM, somatic hypermutation; AID, activation-induced cytidine deaminase; WT, wild type; CSR, class switch recombination; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; HIGM-2, hyper-IgM syndrome; MI, mutability index; nt, nucleotide(s). is characterized by an exceptionally high mutation rate ∼10-3 to 10-4 per base pair per cell division within the V(D)J rearranged Ig genes (1Rajewsky K. Forster I. Cumano A. Science. 1987; 238: 1088-1094Crossref PubMed Scopus (296) Google Scholar). CSR is a unique region specific event in which the donor switch region upstream of the IgH Cμ exon (coding for an IgM antibody) recombines with one of the switch regions that is 5′ to each of the downstream C exons, such as Cγ, Cα, or Cϵ, producing IgG, IgA, or IgE antibody isotypes, respectively (2Stavnezer J. Curr. Top. Microbiol. Immunol. 2000; 245: 127-168PubMed Google Scholar). Typically, under tight transcriptional control, AID is induced in activated B cells within germinal centers, causing deamination of cytosine residues in the variable and switch regions (V- and S-regions, respectively) of transcribed Ig loci. Deaminated DNA is subsequently replicated or repaired by different cellular repair mechanisms to give rise to diversified isotype-switched and antigen-specific high affinity antibodies (for reviews, see Refs. 3Goodman M.F. Scharff M.D. Romesberg F.E. Adv. Immunol. 2007; 94: 127-155Crossref PubMed Scopus (33) Google Scholar, 4Di Noia J.M. Neuberger M.S. Annu. Rev. Biochem. 2007; 76: 1-22Crossref PubMed Scopus (779) Google Scholar, 5Muramatsu M. Nagaoka H. Shinkura R. Begum N.A. Honjo T. Adv. Immunol. 2007; 94: 1-36Crossref PubMed Scopus (99) Google Scholar). Biochemical studies have shown that purified native B-cell AID or recombinant AID protein, expressed in Sf9 insect cells or Escherichia coli, deaminates C residues on ssDNA but not dsDNA, single-stranded RNA, or DNA/RNA hybrid molecules (6Bransteitter R. Pham P. Scharff M.D. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4102-4107Crossref PubMed Scopus (574) Google Scholar, 7Chaudhuri J. Tian M. Khoung C. Chua K. Pinaud E. Alt F.W. Nature. 2003; 421: 726-730Crossref Scopus (613) Google Scholar, 8Dickerson S.K. Market E. Besmer E. Papavasiliou F.N. J. Exp. Med. 2003; 197: 1291-1296Crossref PubMed Scopus (387) Google Scholar, 9Sohail A. Klapacz J. Samaranayake M. Ullah A. Bhagwhat A.S. Nucleic Acids Res. 2003; 31: 2990-2994Crossref PubMed Scopus (239) Google Scholar). AID acts processively on ssDNA and has a distinctive deamination specificity, favoring C targets in WRC hot spot motifs (where W represents A or T, and R is purine) while avoiding SYC cold spots (where S represents C or G and Y is pyrimidine) (10Pham P. Bransteitter R. Petruska J. Goodman M.F. Nature. 2003; 423: 103-107Crossref Scopus (544) Google Scholar, 11Bransteitter R. Pham P. Calabrese P. Goodman M.F. J. Biol. Chem. 2004; 279: 51612-51621Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). These intrinsic biochemical properties of AID are likely to be significant determinants of SHM hallmark features in B-cells, favoring mutations at WRC hot spot motifs and broad clonal heterogeneity of mutations within V-regions (12Rogozin I.B. Kolchanov N.A. Biochim. Biophys. Acta. 1992; 1171: 11-18Crossref PubMed Scopus (405) Google Scholar, 13Smith D.S. Creadon G. Jena P.K. Portanova J.P. Kotzin B.L. Wysocki L.J. J. Immunol. 1996; 156: 2642-2652PubMed Google Scholar, 14Dorner T. Foster S.J. Brezinschek H.-P. Lipsky P.E. Immunol. Rev. 1998; 162: 161-171Crossref PubMed Scopus (87) Google Scholar). Transcription is required for both SHM and CSR to occur (15Peters A. Storb U. Immunity. 1996; 4: 57-65Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 16Fukita Y. Jacobs H. Rajewsky K. Immunity. 1998; 9: 105-114Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 17Maizels N. Cell. 1995; 83: 9-12Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Since AID does not appear to act on dsDNA, transcription probably plays an essential role in generating transient ssDNA, its principal substrate. The ability of AID to deaminate C residues on dsDNA undergoing active transcription has been demonstrated in cells and cell-free assays. For example, ectopically expressed AID can induce mutations on highly transcribed target gene in fibroblasts (18Yoshikawa K. Okazaki I.M. Eto T. Kinoshita K. Muramatsu M. Nagaoka H. Honjo T. Science. 2002; 296: 2033-2036Crossref PubMed Scopus (327) Google Scholar) and E. coli (9Sohail A. Klapacz J. Samaranayake M. Ullah A. Bhagwhat A.S. Nucleic Acids Res. 2003; 31: 2990-2994Crossref PubMed Scopus (239) Google Scholar, 19Ramiro A.R. Stavropoulos P. Jankovic M. Nussenzweig M.C. Nat. Immunol. 2003; 4: 452-456Crossref PubMed Scopus (367) Google Scholar), and purified AID expressed in various sources has been shown to act on dC targets on linear dsDNA and closed circular dsDNA model substrates when transcribed by a prokaryotic RNA polymerase (7Chaudhuri J. Tian M. Khoung C. Chua K. Pinaud E. Alt F.W. Nature. 2003; 421: 726-730Crossref Scopus (613) Google Scholar, 10Pham P. Bransteitter R. Petruska J. Goodman M.F. Nature. 2003; 423: 103-107Crossref Scopus (544) Google Scholar, 11Bransteitter R. Pham P. Calabrese P. Goodman M.F. J. Biol. Chem. 2004; 279: 51612-51621Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 20Shen H.M. Storb U. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12997-13002Crossref PubMed Scopus (129) Google Scholar). AID has been shown to interact with human RNA polymerase II (21Nambu Y. Sugai M. Gonda H. Lee C. Katakai T. Agata Y. Yokota Y. Shimizu A. Science. 2003; 302: 2137-2140Crossref PubMed Scopus (222) Google Scholar), although there are currently no biochemical data for AID acting in conjunction with the human transcription machinery to deaminate dsDNA. Native AID isolated from stimulated primary B-cell nuclei is phosphorylated on Ser38 and Tyr184 residues (22Basu U. Chaudhuri J. Alpert C. Dutt S. Ranganath S. Li G. Schrum J.P. Manis J.P. Alt F.W. Nature. 2005; 438: 508-511Crossref PubMed Scopus (221) Google Scholar, 23McBride K.M. Gazumyan A. Woo E.M. Barreto V.M. Robbiani D.F. Chait B.T. Nussenzweig M.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8798-8803Crossref PubMed Scopus (121) Google Scholar), where Ser38 phosphorylation occurs on about 5–15% of total AID protein (23McBride K.M. Gazumyan A. Woo E.M. Barreto V.M. Robbiani D.F. Chait B.T. Nussenzweig M.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8798-8803Crossref PubMed Scopus (121) Google Scholar). Phosphorylation does not appear to be B-cell-specific, since a similar or greater level of Ser38-phosphorylated AID was observed when expressed in 293T and 3T3-NTZ cells (23McBride K.M. Gazumyan A. Woo E.M. Barreto V.M. Robbiani D.F. Chait B.T. Nussenzweig M.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8798-8803Crossref PubMed Scopus (121) Google Scholar). Protein kinase A (PKA) was found to interact with AID (22Basu U. Chaudhuri J. Alpert C. Dutt S. Ranganath S. Li G. Schrum J.P. Manis J.P. Alt F.W. Nature. 2005; 438: 508-511Crossref PubMed Scopus (221) Google Scholar, 24Pasqualucci L. Kitaura Y. Gu H. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 395-400Crossref PubMed Scopus (150) Google Scholar) and appears to be a primary kinase responsible for phosphorylation at Ser38 and possibly at Thr27 residues in vivo and in vitro (22Basu U. Chaudhuri J. Alpert C. Dutt S. Ranganath S. Li G. Schrum J.P. Manis J.P. Alt F.W. Nature. 2005; 438: 508-511Crossref PubMed Scopus (221) Google Scholar, 24Pasqualucci L. Kitaura Y. Gu H. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 395-400Crossref PubMed Scopus (150) Google Scholar). The potential role of phosphorylation of AID in SHM and CSR was deduced primarily from studies of AID mutations at phosphorylated residues Thr27, Ser38, and Tyr184. A phosphorylation-defective mutant, Y184A, did not affect CSR (22Basu U. Chaudhuri J. Alpert C. Dutt S. Ranganath S. Li G. Schrum J.P. Manis J.P. Alt F.W. Nature. 2005; 438: 508-511Crossref PubMed Scopus (221) Google Scholar). In contrast, a mutation at S38A reduced CSR dramatically to no more than 20% of the wild type (22Basu U. Chaudhuri J. Alpert C. Dutt S. Ranganath S. Li G. Schrum J.P. Manis J.P. Alt F.W. Nature. 2005; 438: 508-511Crossref PubMed Scopus (221) Google Scholar, 24Pasqualucci L. Kitaura Y. Gu H. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 395-400Crossref PubMed Scopus (150) Google Scholar) or moderately (35–80%) (23McBride K.M. Gazumyan A. Woo E.M. Barreto V.M. Robbiani D.F. Chait B.T. Nussenzweig M.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8798-8803Crossref PubMed Scopus (121) Google Scholar, 25Shinkura R. Okazaki I.M. Muto T. Begum N.A. Honjo T. Adv. Exp. Med. Biol. 2007; 596: 71-81Crossref PubMed Scopus (10) Google Scholar) in ex vivo stimulated B-cells. AID S38A caused a significant decrease of SHM in B-cells (23McBride K.M. Gazumyan A. Woo E.M. Barreto V.M. Robbiani D.F. Chait B.T. Nussenzweig M.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8798-8803Crossref PubMed Scopus (121) Google Scholar) and SHM and gene conversion in chicken DT40 cells (26Chatterji M. Unniraman S. McBride K.M. Schatz D.G. J. Immunol. 2007; 179: 5274-5280Crossref PubMed Scopus (29) Google Scholar). Phosphorylated Ser38 was reported to be required for interaction with the 32-kDa subunit of RPA (replication protein A) (27Chaudhuri J. Khuong C. Alt F.W. Nature. 2004; 430: 992-998Crossref PubMed Scopus (326) Google Scholar). AID S38A did not interact with RPA; nor did it catalyze deamination of C on dsDNA in a model T7 RNA polymerase transcription system (22Basu U. Chaudhuri J. Alpert C. Dutt S. Ranganath S. Li G. Schrum J.P. Manis J.P. Alt F.W. Nature. 2005; 438: 508-511Crossref PubMed Scopus (221) Google Scholar, 27Chaudhuri J. Khuong C. Alt F.W. Nature. 2004; 430: 992-998Crossref PubMed Scopus (326) Google Scholar). There are data, however, indicating a lesser role of Ser38 phosphorylation in regulating AID activity. Zebrafish AID, which lacks a serine at a position corresponding to human or mouse Ser38, is fully active in supporting SHM and CSR (28Barreto V.M. Pan-Hammarstrom Q. Zhao Y. Hammarstrom L. Misulovin Z. Nussenzweig M.C. J. Exp. Med. 2005; 202: 733-738Crossref PubMed Scopus (92) Google Scholar, 29Wakae K. Magor B.G. Saunders H. Nagaoka H. Kawamura A. Kinoshita K. Honjo T. Muramatsu M. Int. Immunol. 2006; 18: 41-47Crossref PubMed Scopus (75) Google Scholar) and gene conversion (26Chatterji M. Unniraman S. McBride K.M. Schatz D.G. J. Immunol. 2007; 179: 5274-5280Crossref PubMed Scopus (29) Google Scholar). Recombinant AID expressed in Sf9 cells and E. coli was found to be active on transcribed dsDNA substrates in the absence of RPA (9Sohail A. Klapacz J. Samaranayake M. Ullah A. Bhagwhat A.S. Nucleic Acids Res. 2003; 31: 2990-2994Crossref PubMed Scopus (239) Google Scholar, 10Pham P. Bransteitter R. Petruska J. Goodman M.F. Nature. 2003; 423: 103-107Crossref Scopus (544) Google Scholar, 11Bransteitter R. Pham P. Calabrese P. Goodman M.F. J. Biol. Chem. 2004; 279: 51612-51621Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 30Shen H.M. Ratnam S. Storb U. Mol. Cell Biol. 2005; 25: 10815-10821Crossref PubMed Scopus (52) Google Scholar, 31Besmer E. Market E. Papavasiliou F.N. Mol. Cell Biol. 2006; 26: 4378-4385Crossref PubMed Scopus (60) Google Scholar). Thus, although it is clear that a minority of AID molecules are phosphorylated at Ser38, it is considerably less clear how the biochemical behavior of AID is influenced by either the location of phosphorylated residues or whether or not a particular residue is phosphorylated. The roles of phosphorylation on the properties of AID in vitro are amenable to a comparative biochemical analysis using wild type (WT) AID and phosphorylation-defective mutants. Here we have investigated how replacing individual “phosphorylation-active” Ser residues influences AID specific activity, processivity, deamination specificity, transcriptional-dependent deamination, including its interactions with RPA, and mutational spectra. One such mutant, S43P, has been identified in humans diagnosed with hyper-IgM (HIGM-2) syndrome (32Zhu Y. Nonoyama S. Morio T. Muramatsu M. Honjo T. Mizutani S. J. Med. Dent. Sci. 2003; 50: 41-46PubMed Google Scholar), characterized by the absence of CSR. A comparison of deamination motif specificities favored by S43P in relation to WT AID suggests a possible connection of AID deamination specificity with human immunodeficiency disease. Enzymes and Substrates—Mutant AID proteins (S38A, S38D, S41A, S41D, S43A, and S43P) were constructed by site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene) using the pAcG2T-AID vector (6Bransteitter R. Pham P. Scharff M.D. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4102-4107Crossref PubMed Scopus (574) Google Scholar, 10Pham P. Bransteitter R. Petruska J. Goodman M.F. Nature. 2003; 423: 103-107Crossref Scopus (544) Google Scholar) as the template. Recombinant baculoviruses encoding WT and mutant AID were generated according to the recommended protocol (BD Bioscience). WT and mutant GST-AID proteins were expressed and purified as described previously (10Pham P. Bransteitter R. Petruska J. Goodman M.F. Nature. 2003; 423: 103-107Crossref Scopus (544) Google Scholar, 11Bransteitter R. Pham P. Calabrese P. Goodman M.F. J. Biol. Chem. 2004; 279: 51612-51621Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), with an additional of Halt phosphatase inhibitor mixture (Pierce) in the lysis buffer. AID proteins were dialyzed in a buffer containing 20 mm Tris-HCl (pH 7.5), 25 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, and 10% glycerol and stored at -80 °C. E. coli single-stranded binding protein and recombinant human RPA were overexpressed in E. coli and purified according to published protocols (33Lohman T.M. Green J.M. Beyer R.S. Biochemistry. 1986; 25: 21-25Crossref PubMed Scopus (194) Google Scholar, 34Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). T7 RNA polymerase was purchased from Promega, and ultrapure NTP was purchased from Amersham Biosciences. The active, recombinant catalytic subunit of human protein kinase A was purchased from Calbiochem. M13mp2 gapped DNA and M13mp2T7 covalently closed circular dsDNA substrates were prepared as described (10Pham P. Bransteitter R. Petruska J. Goodman M.F. Nature. 2003; 423: 103-107Crossref Scopus (544) Google Scholar, 11Bransteitter R. Pham P. Calabrese P. Goodman M.F. J. Biol. Chem. 2004; 279: 51612-51621Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Mass Spectrometry Analysis—Approximately 20 μg of purified AID were reduced with 5 mm dithiothreitol in the presence of 1% SDS and subsequently alkylated with 20 mm iodoacetamide. Protein was then precipitated by the addition of 3 volumes of a solution containing 50% acetone, 49.9% ethanol, and 0.1% acetic acid; the sample was kept on ice for 15 min and then centrifuged for 10 min at maximum speed on a bench top centrifuge. The pellet containing precipitated AID protein was resuspended in 40 μl of 8 m urea, 100 mm Tris-HCl, pH 8.0. The suspension was then diluted by adding 160 μl of 150 mm NaCl, 50 mm Tris-HCl, pH 8.0, and the protein was digested with 2 μg of trypsin (Promega) overnight. After digestion, 0.4% trifluoroacetic acid was added, and the peptides were desalted using a 50-mg C18-Sep-Pak cartridge (Waters). Phosphopeptides were purified by IMAC as previously described (35Smolka M.B. Albuquerque C.P. Chen S.H. Schmidt K.H. Wei X.X. Kolodner R.D. Zhou H. Mol. Cell Proteomics. 2005; 4: 1358-1369Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Purified phosphopeptides were analyzed by microcapillary-liquid chromatography-electrospray ionization-tandem mass spectrometry on a Thermo Finnigan LTQ quadrupole ion trap mass spectrometer, as described (35Smolka M.B. Albuquerque C.P. Chen S.H. Schmidt K.H. Wei X.X. Kolodner R.D. Zhou H. Mol. Cell Proteomics. 2005; 4: 1358-1369Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). For data analysis, the SEQUEST (version 3.4 beta 2) program running on a Sorcerer system (SageN, San Jose, CA) was used for peptide identification. A data base search was performed using a sub-data base consisting of 50 budding yeast proteins in addition to the human AID sequence. The following variable modifications were considered: +80 Da (phosphorylation) for serine, threonine and tyrosine residues; +16 Da (oxidation) for methionine residues. Up to four variable modifications were allowed per peptide, and the peptide mass tolerance used was 3 Da. A semitryptic restriction was applied, and only the top-matched peptides with a probability score above 0.9 were subsequently considered for close inspection. Each MS/MS spectrum that led to a phosphopeptide identification was manually verified to confirm that all significant ions were accounted for and then validated. Measurements of Deamination-specific Activity on ssDNA—Specific activities of WT AID and phosphorylation mutants were measured using 32P-labeled 36-nt ssDNA 5′-AGAAAAGGGGAAAGCAAAGAGGAAAGGTGAGGAGGT-3′. Reactions were carried out in a buffer containing 50 mm HEPES (pH 7.5), 1 mm dithiothreitol, 10 mm MgCl2 in the presence of 500 fmol of the substrate DNA, 200 fmol of recombinant GST-AID (Sf9-expressed) or 6400 fmol of E. coli expressed GST-AID, and 20 ng of RNase A. Following incubation at 37 °C for 5 min, the reactions were quenched by a double extraction with phenol/chloroform/isoamyl alcohol (25:24:1), and the deamination product was analyzed as described previously (6Bransteitter R. Pham P. Scharff M.D. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4102-4107Crossref PubMed Scopus (574) Google Scholar). Specific activities were calculated as amount (fmol) of deaminated substrate/min/μg of enzyme. Deamination Activity of WT and Mutant AID on Transcribed dsDNA—Transcription-dependent AID deamination on the nontranscribed strand of dsDNA was measured using a linear dsDNA, undergoing active transcription by T7 RNA polymerase (10Pham P. Bransteitter R. Petruska J. Goodman M.F. Nature. 2003; 423: 103-107Crossref Scopus (544) Google Scholar). In a typical reaction (50 μl), GST-AID (7 pmol of Sf9-expressed WT or mutant AID or 50 pmol of E. coli-expressed AID), RNase A (200 ng), dsDNA substrate (1 pmol), and T7 RNA polymerase (1 μl) in a reaction buffer containing HEPES (50 mm, pH 7.5), dithiothreitol (5 mm), MgCl2 (10 mm), and NTPs (250 μm) were incubated at 37 °C for 30 min. E. coli SSB or human RPA (3, 6, or 12 pmol), if present, were added to the reactions as indicated. The reactions were stopped by extracting twice with phenol/chloroform/isoamyl alcohol (25:24:1). The deaminated products were analyzed using a primer elongation-dideoxynucleotide termination assay (10Pham P. Bransteitter R. Petruska J. Goodman M.F. Nature. 2003; 423: 103-107Crossref Scopus (544) Google Scholar). Thermo Sequenase (U.S. Biochemical Corp.) was used to extend an 18-mer 32P-labeled primer annealed to the target strand in the presence of three dNTPs and either ddATP or ddGTP (80 μm each). The reactions were carried out for seven cycles (95 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min) and terminated by adding an equal volume of stop solution containing 95% formamide and 20 mm EDTA. The reaction products were resolved by 19% polyacrylamide denaturing gel electrophoresis and analyzed by phosphorimaging. Deamination efficiencies were calculated from extension reactions with the ddA mix as a ratio of the band intensity opposite the converted U template site compared with integrated band intensities opposite and beyond the C template site. The efficiencies were also calculated from extension reactions with ddG as a ratio of integrated band intensities beyond the C template site to the integrated band intensities opposite and beyond the C template site. Mutation Analysis of AID-targeted C Deamination in Vitro—Deamination specificities of WT and mutant AID were measured using the following reaction conditions: 30-μl volume, 50 mm HEPES (pH 7.5), dithiothreitol (1 mm), MgCl2 (10 mm), gapped DNA (500 ng), RNase A (200 ng), and WT or mutant AID (50 to 100 ng of Sf9-expressed AID or 1 μg of E. coli-expressed AID). Following incubations for 2.5, 5, and 10 min at 37 °C, the reactions were quenched by a double extraction with phenol/chloroform/isoamyl alcohol (25:24:1). Conversions of C → U on the DNA substrate were detected as white or light blue plaques, indicating C → T mutations in a lacZα target gene after transfection into uracil glycosylase-deficient (ung-) E. coli, as previously described (10Pham P. Bransteitter R. Petruska J. Goodman M.F. Nature. 2003; 423: 103-107Crossref Scopus (544) Google Scholar). WT and mutant AID deamination spectra were compared using χ2 tests with the null hypothesis that the distribution of deaminations at the nucleotide positions is the same for two enzymes. We consider a contingency table where the two columns are the WT and mutant AID, the rows are the nucleotide sites, and the numbers in the cells are the deaminations corresponding to that column and row. In order to deal with the problem of small denominators, we only consider those sites for which the sum of the deaminations for the two enzymes is 5 or greater (we have tried other thresholds and reached the same conclusions). In order to determine whether a motif is deaminated differently by the WT or mutant enzymes, we performed the following test. For each WT or mutant enzyme (S38A, S41A, S43A, or S43P), we calculated the “site mutability index” by dividing the number of deaminations at each site by the total number of deaminations. For the set of sites sharing the motif, we compared the WT index with each of the four mutant AID indexes by the paired Wilcox test. We used all four mutants, since their spectra are similar to each other (but not the WT), and there are a relatively small number of sites for each motif. Since we repeated this test for each of the 16 motifs, the p values in Table 2 have been corrected for multiple tests. The reported value is the probability that if 16 independent tests have been performed, then at least one will be as extreme as observed: c = 1 - (1 - p)16, where c is the correction of p from the Wilcox test.TABLE 2Three-nucleotide motif MI for WT and mutant AID (S38A and S43P) at 2.5 min The MI is defined as the number of times a given trinucleotide motif within a segment of DNA contains a mutation, divided by the number of times the oligonucleotide would be expected to be mutated for a mechanism with no sequence bias.MotifMIWTS38AS43Pp valueap values were calculated by the paired Wilcox test (see “Experimental Procedures”)Hot spots AAC2.51.51.60.41 AGC1.32.12.00.06 TAC3.42.02.20.22 TGC2.62.22.60.99 MI average (WRC)2.51.92.1Cold spots CCC0.280.520.440.65 CTC0.450.760.530.99 GCC0.050.180.050.07 GTC0.320.670.000.99 MI average (SYC)0.280.530.26Intermediates ACC1.20.320.350.02 ATC1.00.630.710.97 CAC1.40.910.960.31 CGC0.51.31.40.004 GAC0.240.50.350.79 GGC0.230.781.00.0001 TCC0.481.11.10.75 TTC0.530.710.710.46 MI average0.700.780.82a p values were calculated by the paired Wilcox test (see “Experimental Procedures”) Open table in a new tab Processivity Deamination Analysis of WT and Mutant AID—An 85-nt ssDNA substrate with two identical hot spot AGC motifs, containing a fluorescein tag located between the two motifs, was used. Deamination reactions (45-μl volume) were carried out in the presence of 15 pmol of ssDNA substrate, 5–15 pmol of WT or mutant GST-AID, and 200 ng of RNase A for 2.5, 5, and 10 min. After treatment with 4 units of uracil DNA glycosylase (New England Biolabs) and alkali, the deamination products were separated on a 16% denaturing polyacrylamide gel, visualized, and quantified using a FX fluorescence scanner (Bio-Rad). Analysis of processive deamination by a single WT and mutant AID enzyme was carried out as described (36Chelico L. Pham P. Calabrese P. Goodman M.F. Nat. Struct. Mol. Biol. 2006; 13: 392-399Crossref PubMed Scopus (241) Google Scholar, 37Pham P. Chelico L. Goodman M.F. DNA Repair (Amst.). 2007; 6: 689-692Crossref PubMed Scopus (24) Google Scholar). The single deamination rates are approximately equal at the 5′-C and 3′-C target motifs. The correlated double deamination efficiency is calculated as the probability that a single AID molecule deaminates both 5′ and 3′ target motifs on one ssDNA substrate during a single enzyme-DNA encounter. Human AID Expressed in Sf9 Cells Is Partially Phosphorylated at Ser Residues 38, 41, and 43 and at Thr27—Mass spectral analysis demonstrates that WT recombinant AID expressed in Sf9 insect cells is phosphorylated at four predicted protein kinase motifs; two motifs are the previously identified Ser38 and Thr27 residues (22Basu U. Chaudhuri J. Alpert C. Dutt S. Ranganath S. Li G. Schrum J.P. Manis J.P. Alt F.W. Nature. 2005; 438: 508-511Crossref PubMed Scopus (221) Google Scholar, 24Pasqualucci L. Kitaura Y. Gu H. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 395-400Crossref PubMed Scopus (150) Google Scholar), and two newly identified residues are at Ser41 and Ser43 (Fig. 1). Individual peptide fragments of AID showed that just one of the four residues was phosphorylated. Multiple phosphorylations of individual proteins were not detected despite using an isolation technique that strongly favored the detection of multiply phosphorylated peptide fragments (Fig. 1). These three Ser residues 38, 41, and 43 along with Thr27 are predicted to be phosphorylation sites for protein kinase PKA or CAMK2, protein kinase Cδ, CK1, and PKA, respectively (Web Scansite 2.0, medium stringency scan (38Obenauer J.C. Cantley L.C. Yaffe M.B. Nucleic Acids Res. 2003; 31: 3635-3641Crossref PubMed Scopus (1342) Google Scholar)). Based on the amount of protein used in the analysis, we estimate that the phosphorylated species represents less than a few percent of the total AID. The detection of phosphorylated AID required that the enzyme isolation and purification be carried out in the presence of a mixture of phosphatase inhibitors (see “Experimental Pro" @default.
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• W2023254248 title "Impact of Phosphorylation and Phosphorylation-null Mutants on the Activity and Deamination Specificity of Activation-induced Cytidine Deaminase" @default.
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