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The cytidine deaminase AID exhibits similar functional properties in yeast and mammals
Molecular Immunology 43 (2006) 1481–1484
Short communication
The cytidine deaminase AID exhibits similar functional properties in yeast and mammals Igor B. Rogozin a,∗ , Youri I. Pavlov b,c,d a
National Center for Biotechnology Information NLM, National Institutes of Health, 8600 Rockville Pike, Bldg. 38A/room 5N505A, Bethesda, MD 20894, USA b Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA c Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA d Department of Pathology and Microbiology, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA Received 19 July 2005 Available online 10 October 2005
Abstract A recent work published in Molecular Immunology examined the editing activity of activation-induced deaminase (AID) in yeast (Krause, K., Marcu, K.B., Greeve, J., 2006. The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system. Mol. Immunol.). It was proposed that expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed gene due to the lack of cofactors for AID-induced somatic hypermutation, which are unique to B cells. It was suggested that, on its own, AID does not have an intrinsic specificity for its target sequences. However, it has been shown previously that expression of the human AID gene in yeast was moderately mutagenic in a wild-type strain and highly mutagenic in an ung1 uracil-DNA glycosylase-deficient strain (Mayorov, V.I., Rogozin, I.B., Adkison, L.R., Frahm, C.R., Kunkel T.A., Pavlov Y. I., 2005. Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at G-C pairs in immunoglobulin genes. BMC Immunol. 6, 10; Poltoratsky, V.P., Wilson, S.H., Kunkel, T.A., Pavlov, Y.I., 2004. Recombinogenic phenotype of human activation-induced cytosine deaminase. J. Immunol. 172, 4308–4313). The vast majority of mutations were at G-C pairs. Mutations showed a clear DNA sequence context specificity which resembled the specificity of somatic hypermutation at G-C pairs in immunoglobulin genes and AID mutation specificity in vitro. The inability to detect mutator effects of AID by Krause et al. is likely to be caused by the use of the wild-type yeast strain and a small sample of clones examined for the presence of mutations. In addition, we show that non-uniformity of the mutation hotspot distribution is a factor potentially decreasing the chances of detecting mutations. © 2005 Elsevier Ltd. All rights reserved. Keywords: Somatic hypermutation; DNA context; Polynucleotide (deoxy)cytidine deaminase; Mutation hotspots; Mutation frequency
1. Introduction Antibody genes are diversified by somatic hypermutation (SHM), gene conversion (IGC), and class-switch recombination (CSR) (Honjo et al., 1981). All three processes are initiated by activation-induced deaminase (AID) (Honjo et al., 2004; Neuberger et al., 2003). Initially, AID was thought to act in mutagenesis and recombination via its RNA editing activity. Specifically, the RNA-editing model postulated that AID edits the pre-mRNA for a nicking endonuclease that initiates SHM, IGC and CSR (Kinoshita and Honjo, 2001). AID is homologous to the RNA-editing enzyme APOBEC1, which deaminates
∗
Corresponding author. Tel.: +1 301 594 8079; fax: +1 301 435 7794. E-mail address: [email protected] (I.B. Rogozin).
0161-5890/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2005.09.002
cytosine at position 6666 in ApoB100 mRNA and seemingly has no role in immunity. AID was reported to deaminate cytidine, and shuttles between the nucleus and cytoplasm similarly to APOBEC1. A different, DNA deamination hypothesis suggests that AID deaminates cytosine in DNA directly and that uracil generated in this reaction triggers downstream reactions leading to genetic instability (Neuberger et al., 2003). The DNA deamination hypothesis of AID function is supported by many direct observations (Pham et al., 2005). However, it is possible that AID has a dual function in B cells, i.e., edits both DNA and RNA. AID is able to induce genome-wide hypermutation in a variety of mammalian non-B cells suggesting that this protein is the most critical B cell specific component required for the induction of mutations (Martin et al., 2002; Wang et al., 2004; Yoshikawa et al., 2002). The global mutation rates measured in several
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reporters by PCR-based technique were estimated to be in the range of 5 × 10−3 –10−5 per nucleotide in mammalian cells expressing AID. Such a level of mutagenesis would generate from thousands to millions of new mutations in the euchromatic part of the genome during each division. When the mutator effect of AID was estimated by the standard genetic method in CHO cells expressing AID, it was found that reversion frequency was increased only 15-fold in comparison to control cells (Martin et al., 2002). AID can also induce mutations when expressed in E. coli (Beale et al., 2004; Petersen-Mahrt et al., 2002). The estimated rates were 10−9 –10−10 per nucleotide in a wild-type strain and an order of magnitude higher in cells unable to excise uracil from DNA. The enhancement of mutagenesis in a uracil-DNA glycosylase-deficient ung1 strain suggests that the deamination of cytosine to uracil in DNA is the cause of mutations induced by AID. Almost all mutations arising upon expression of AID were G-A to A-T transitions, consistent with the DNA deamination model. It has been shown that AID is indeed capable of deaminating single-stranded and supercoiled, double-stranded DNA (Pham et al., 2003, 2005; Shen and Storb, 2004). The DNA sequence context specificity of this reaction resembled the specificity of SHM at G-C pairs (DGYW/WRCH motifs; mutating G-C are underlined, D = A/T/G, H = C/A/T, Y = T/C, R = A/G, W = A/T) (Rogozin and Diaz, 2004) and AID mutation specificity in vitro (GYW/WRC motifs) (Pham et al., 2003, 2005). Therefore, eukaryotic cell-specific components may be necessary for delivery of AID to regions undergoing SHM, but not for AID-induced mutagenesis per se. In a recent paper published in Molecular Immunology, Krause et al. analyzed the RNA and DNA editing activity of AID in yeast using a selectable marker and concluded that the expression of AID in yeast, as opposed to APOBEC1, did not lead to RNA editing and was not sufficient for the generation of point mutations in a highly transcribed gene (Krause et al., 2006). They speculated that cofactors for AID-induced somatic hypermutation, that are present in B cells and a variety of mammalian non-B cells, are missing in yeast (Krause et al., 2006). Here, we present arguments that this conclusion, most likely, is due to lack of power of the approach used by Krause et al. (2006). 2. Materials and methods The collection of mutations in the CAN1 gene induced by the expression of AID in yeast has been described before (Mayorov et al., 2005); and mutations from wild-type and ung1 strains were merged into one spectrum. Spectra of somatic mutations in an artificially synthesized EPS sequence were generated by Storb and co-workers (Kim et al., 1999; Storb et al., 1998). Mutations in the GFP gene induced by the expression of AID in murine fibroblasts were generated by Yoshikawa and co-workers (Yoshikawa et al., 2002). Mutation hotspots were defined using a threshold (Th ) for the number of mutations at a site. The threshold is established by analyzing the frequency distribution derived from a mutation spectrum using the CLUSTERM program (www.itb.cnr.it/webmutation/) (Glazko et al., 1998). Briefly, this program decomposes a mutation spectrum into several homogeneous classes of sites, with each class approximated
by a binomial distribution. Variations in mutation frequencies among sites of the same class are random by definition (mutation probability is the same for all sites within a class), but differences between classes are statistically significant. Each site has a probability P(C) to be assigned to class C. A class with the highest mutation frequency is called the hotspot class. Sites with P(Chotspot ) > 0.95 are be assigned to the hotspot class Chotspot and defined as hotspot sites. This approach ensures that the assignment is statistically significant and robust [see Rogozin et al. (2001) for a detailed discussion of this approach and problems associated with its application]. 3. Results and discussion We studied the genetic consequences of AID expression in yeast using well-established, precise mutation assays available for this organism (Mayorov et al., 2005; Poltoratsky et al., 2004). We found that the expression of the human AID gene was moderately mutagenic in the wild-type strain (mutation rates, ranging from 10−8 to 3 × 10−7 per reporter, were increased up to eight-fold in the presence of AID, depending on the marker) and highly mutagenic (up to 46-fold) in the ung1 uracil-DNA glycosylase-deficient strain (Mayorov et al., 2005). The increase in the mutation rate observed in the yeast wild-type strain is similar to the increase found in CHO cells expressing AID (Martin et al., 2002). The number of detectable positions in the reporters used is not defined, therefore we can only make a safe estimate of the maximum mutation rate per nucleotide. For wild-type strain expressing AID, it is 3.7 × 10−6 . The majority of mutations were at G-C pairs, and DGYW/WRCH motifs (Rogozin and Diaz, 2004) were preferential sites of mutations (Table 1). It was concluded that the intrinsic substrate specificity of AID itself is the primary determinant of mutation hotspots at G-C base pairs during SHM (Mayorov et al., 2005). There are two possible explanations for the discrepancy between the conclusions of these two studies (Mayorov et al., 2005; Krause et al., 2006). The frequency of mutations induced by AID estimated by classical methods is lower in comparison to PCR-based estimates of AID-induced mutation rates in mammalian cells (see above). Most likely, sequencing of 6 × 103 bases by (Krause et al., 2006) failed to detect any mutations in yeast due to the use of wild-type strains (capable of removing Table 1 Mutation hotspots induced by the expression of AID in yeast Position
Sequence
Number of substitutions
DGYW/WRCH variant (D = A/T/G, H = T/A/C)
238 268 299 896 980 1166 1392 1426
GTACAGA AAGCAAA GTGGTAC AAGGTAC TCCGTAT CTGCCGC ATGGTTA ATGCAAG
6 9 5 4 8 4 5 5
WRCA WRCA GGYW GGYW – WRCC GGYW WRCA
The data was taken from (Mayorov et al., 2005), the hotspot threshold Th is four mutations.
I.B. Rogozin, Y.I. Pavlov / Molecular Immunology 43 (2006) 1481–1484 Table 2 DNA context of mutations induced by the expression of AID in murine fibroblasts Start/end of motifs
Substitutions
Hotspots
AGCA 94–97 329–332 560–563 641–643 697–700 715–718
2G → A, 4G → C, 1G → T 1G → A, 1C → T 1G → T, 1C → G 1C → T, 1C → A 3G → A, 1G → T 2G → C, 5C → T
Yes No No No No Yes
TGCA 232–235 296–299 620–623 843–846
2G → A 1G → T, 1G → C, 1C → T 1G → A, 2C → T No mutations
No No No No
AGCT 21–24 107–110 140–143 212–215 248–251 464–467 512–515 623–626 797–800 825–828 831–834
2G → A 2G → C, 3C → T, 1C → A 1G → A, 1G → T, 6G → C, 4C → T 1G → A, 1G → C, 7C → T, 3C → G 5G → A, 1G → T, 1G → C, 2C → T, 1C → G 1G → C, 3C → T 2G → A, 1G → T, 2C → T, 1C → G 3G → A, 1G → C, 1C → T, 1C → G 2G → A, 2G → C 3G → A, 1G → T, 1G → C, 2C → T, 2C → G 2C → T
No No Yes Yes Yes No No No No No
TGCT 301–304 671–674 752–755 930–933
1G → A, 1G → T, 2C → T, 2C → G 5G → A, 2G → T, 4C → T 1G → C No mutations
No Yes No No
The data was taken from (Yoshikawa et al., 2002). Only GC-containing DGYW/WRCH motifs are shown, the hotspot threshold Th is five mutations.
uracil from DNA) and the low resolution of analysis of putative mutations (estimated maximum rate of AID-induced mutations is 3 per 106 nucleotides, see above). When a selective test for mutations was used by Krause and co-workers, the detection limit was apparently low too, less that 1 mutant per 5 × 105 yeast colonies (Krause et al., 2006). In addition, it is possible that the target gene selected by Krause and co-workers had low mutability by DNA deamination (Krause et al., 2006). As correctly pointed out by Krause et al., the sequence of Gal4-VH4 contains several DGYW/WRCH motifs, mutations C → T are detectable in two sequences,
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AGCA and TGCA (Krause et al., 2006). These sequences are mutable in SHM (Rogozin and Diaz, 2004). However, mutable motifs alone are not sufficient for the emergence of hotspots (Rogozin and Pavlov, 2003). We analyzed the distribution of mutations in AGCA, TGCA, AGCT and TGCT sequences induced by expression of AID in murine fibroblasts (Yoshikawa et al., 2002). Only six of the 24 mutable sequences contain mutation hotspots (Table 2). The distribution of mutation hotspot across the GFP gene did not show any obvious pattern: four hotspots are located in the beginning of the GFP gene and two are located towards the end of the gene, and long regions of the gene contain no hotspots (Table 2). Non-uniformity of the hotspot distribution may be also detected for closely located mutable motifs as illustrated by the distribution of somatic mutations across mutable AGCT sequences in an artificially synthesized EPS sequence inserted into an immunoglobulin gene (Fig. 1) (Kim et al., 1999; Storb et al., 1998). The EPS sequence contains AGCT sequences repeated six times, respectively (PA-PF monomeric units in Fig. 1). The number of mutations at G-C bases within AGCT sites varied from four (the PF monomer) to 21 (the PD monomer). Two significantly different classes of AGCT motifs were revealed by the CLUSTERM program (Glazko et al., 1998). The hotspot class includes PA, PB, PC, and PD sequences (Fig. 1). Another class consists of PE and PF sequences which have significantly lower frequencies of mutations. This result shows that a significant heterogeneity of the mutation rate exists even in monotonously repeated AGCT motifs located as far as 600 nucleotides away from the transcription start (Fig. 1). These two examples show that mutation hotspots are not equivalent to mutable motifs. The emergence of hotspots is a complex process that depends on multiple factors. Thus, some mutable motifs are cold spots in an unfavorable global sequence context (Rogozin and Pavlov, 2003). This might add further explanation for the lack of mutations in two DGYW/WRCH motifs observed by Krause and co-workers (Krause et al., 2006). Another possible explanation for the absence of mutations in samples analyzed by Krause and co-workers may be the use of plasmids while other authors studied mutations in chromosomal genes. This explanation is less likely because the detection of recombination events by Krause and co-workers (Krause et al., 2006) suggests that AID in these experiments deaminated the plasmid DNA; it has been demonstrated previously that the expression of AID leads to the induction of mitotic intragenic recombination between chromosomal alleles in uracil-DNA glycosylase-proficient yeast (Poltoratsky et al., 2004).
Fig. 1. Somatic hypermutation spectrum in an artificially synthesized EPS sequence (Kim et al., 1999; Storb et al., 1998). The AGCT mutable motifs are underlined. Position numbers refer to the transcription start site.
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The results of Mayorov et al. (2005) are consistent with the hypothesis that AID-mediated deamination of DNA is a major cause of mutations at G-C base pairs in immunoglobulin genes during SHM. A general pattern of mutations and frequencies of AID-induced substitutions in yeast is similar to the targeting of somatic mutation to the DGYW/WRCH mutable motifs, which are highly specific for SHM in mammals (Rogozin and Diaz, 2004). This indicates that the intrinsic substrate specificity of AID itself is the primary determinant of mutation hotspots at GC base pairs during SHM. Quantitative aspects of the mutability of those genomes exposed to AID vary from species to species and, in multicellular organisms, depend on cell types. Discrepancies among estimates of mutability in the same organisms may be explained by the difference in the genetic background and experimental approaches employed for the analysis of mutagenesis. References Beale, R.C., Petersen-Mahrt, S.K., Watt, I.N., Harris, R.S., Rada, C., Neuberger, M.S., 2004. Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo. J. Mol. Biol. 337, 585–596. Glazko, G.V., Milanesi, L., Rogozin, I.B., 1998. The subclass approach for mutational spectrum analysis: application of the SEM algorithm. J. Theor. Biol. 192, 475–487. Honjo, T., Muramatsu, M., Fagarasan, S., 2004. AID: how does it aid antibody diversity? Immunity 20, 659–668. Honjo, T., Nakai, S., Nishida, Y., Kataoka, T., Yamawaki-Kataoka, Y., Takahashi, N., Obata, M., Shimizu, A., Yaoita, Y., Nikaido, T., Ishida, N., 1981. Rearrangements of immunoglobulin genes during differentiation and evolution. Immunol. Rev. 59, 33–67. Kim, N., Bozek, G., Lo, J.C., Storb, U., 1999. Different mismatch repair deficiencies all have the same effects on somatic hypermutation: intact primary mechanism accompanied by secondary modifications. J. Exp. Med. 190, 21–30. Kinoshita, K., Honjo, T., 2001. Linking class-switch recombination with somatic hypermutation. Nat. Rev. Mol. Cell. Biol. 2, 493–503. Krause, K., Marcu, K.B., Greeve, J., 2006. The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system. Mol. Immunol. 43, 295–307.
Martin, A., Bardwell, P.D., Woo, C.J., Fan, M., Shulman, M.J., Scharff, M.D., 2002. Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas. Nature 415, 802–806. Mayorov, V.I., Rogozin, I.B., Adkison, L.R., Frahm, C.R., Kunkel, T.A., Pavlov, Y.I., 2005. Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at G-C pairs in immunoglobulin genes. BMC Immunol. 6, 10. Neuberger, M.S., Harris, R.S., Di Noia, J., Petersen-Mahrt, S.K., 2003. Immunity through DNA deamination. Trends Biochem. Sci. 28, 305– 312. Petersen-Mahrt, S.K., Harris, R.S., Neuberger, M.S., 2002. AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–103. Pham, P., Bransteitter, R., Petruska, J., Goodman, M.F., 2003. Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424, 103–107. Pham, P., Bransteitter, R., Goodman, M.F., 2005. Reward versus risk: DNA cytidine deaminases triggering immunity and disease. Biochemistry 44, 2703–2715. Poltoratsky, V.P., Wilson, S.H., Kunkel, T.A., Pavlov, Y.I., 2004. Recombinogenic phenotype of human activation-induced cytosine deaminase. J. Immunol. 172, 4308–4313. Rogozin, I.B., Diaz, M., 2004. Cutting edge: DGYW/WRCH is a better predictor of mutability at G:C bases in Ig hypermutation than the widely accepted RGYW/WRCY motif and probably reflects a two-step activation-induced cytidine deaminase-triggered process. J. Immunol. 172, 3382–3384. Rogozin, I.B., Kondrashov, Glazko, G.V., 2001. Use of mutation spectra analysis software. Hum. Mutat. 17, 83–102. Rogozin, I.B., Pavlov, Y.I., 2003. Theoretical analysis of mutational hotspots and their DNA sequence context specificity. Mutat. Res. 544, 65–85. Shen, H.M., Storb, U., 2004. Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled. Proc. Natl. Acad. Sci. USA 101, 12997–13002. Storb, U., Klotz, E.L., Hackett, J., Kage, K., Bozek, G., Martin, T.E., 1998. A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript. J. Exp. Med. 188, 689–698. Wang, C.L., Harper, R.A., Wabl, M., 2004. Genome-wide somatic hypermutation. Proc. Natl. Acad. Sci. USA 101, 7352–7356. Yoshikawa, K., Okazaki, I.M., Eto, T., Kinoshita, K., Muramatsu, M., Nagaoka, H., Honjo, T., 2002. AID enzyme-induced hypermutation in an actively transcribed gene in fibroblasts. Science 296, 2033– 2036.
Short communication
The cytidine deaminase AID exhibits similar functional properties in yeast and mammals Igor B. Rogozin a,∗ , Youri I. Pavlov b,c,d a
National Center for Biotechnology Information NLM, National Institutes of Health, 8600 Rockville Pike, Bldg. 38A/room 5N505A, Bethesda, MD 20894, USA b Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA c Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA d Department of Pathology and Microbiology, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA Received 19 July 2005 Available online 10 October 2005
Abstract A recent work published in Molecular Immunology examined the editing activity of activation-induced deaminase (AID) in yeast (Krause, K., Marcu, K.B., Greeve, J., 2006. The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system. Mol. Immunol.). It was proposed that expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed gene due to the lack of cofactors for AID-induced somatic hypermutation, which are unique to B cells. It was suggested that, on its own, AID does not have an intrinsic specificity for its target sequences. However, it has been shown previously that expression of the human AID gene in yeast was moderately mutagenic in a wild-type strain and highly mutagenic in an ung1 uracil-DNA glycosylase-deficient strain (Mayorov, V.I., Rogozin, I.B., Adkison, L.R., Frahm, C.R., Kunkel T.A., Pavlov Y. I., 2005. Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at G-C pairs in immunoglobulin genes. BMC Immunol. 6, 10; Poltoratsky, V.P., Wilson, S.H., Kunkel, T.A., Pavlov, Y.I., 2004. Recombinogenic phenotype of human activation-induced cytosine deaminase. J. Immunol. 172, 4308–4313). The vast majority of mutations were at G-C pairs. Mutations showed a clear DNA sequence context specificity which resembled the specificity of somatic hypermutation at G-C pairs in immunoglobulin genes and AID mutation specificity in vitro. The inability to detect mutator effects of AID by Krause et al. is likely to be caused by the use of the wild-type yeast strain and a small sample of clones examined for the presence of mutations. In addition, we show that non-uniformity of the mutation hotspot distribution is a factor potentially decreasing the chances of detecting mutations. © 2005 Elsevier Ltd. All rights reserved. Keywords: Somatic hypermutation; DNA context; Polynucleotide (deoxy)cytidine deaminase; Mutation hotspots; Mutation frequency
1. Introduction Antibody genes are diversified by somatic hypermutation (SHM), gene conversion (IGC), and class-switch recombination (CSR) (Honjo et al., 1981). All three processes are initiated by activation-induced deaminase (AID) (Honjo et al., 2004; Neuberger et al., 2003). Initially, AID was thought to act in mutagenesis and recombination via its RNA editing activity. Specifically, the RNA-editing model postulated that AID edits the pre-mRNA for a nicking endonuclease that initiates SHM, IGC and CSR (Kinoshita and Honjo, 2001). AID is homologous to the RNA-editing enzyme APOBEC1, which deaminates
∗
Corresponding author. Tel.: +1 301 594 8079; fax: +1 301 435 7794. E-mail address: [email protected] (I.B. Rogozin).
0161-5890/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2005.09.002
cytosine at position 6666 in ApoB100 mRNA and seemingly has no role in immunity. AID was reported to deaminate cytidine, and shuttles between the nucleus and cytoplasm similarly to APOBEC1. A different, DNA deamination hypothesis suggests that AID deaminates cytosine in DNA directly and that uracil generated in this reaction triggers downstream reactions leading to genetic instability (Neuberger et al., 2003). The DNA deamination hypothesis of AID function is supported by many direct observations (Pham et al., 2005). However, it is possible that AID has a dual function in B cells, i.e., edits both DNA and RNA. AID is able to induce genome-wide hypermutation in a variety of mammalian non-B cells suggesting that this protein is the most critical B cell specific component required for the induction of mutations (Martin et al., 2002; Wang et al., 2004; Yoshikawa et al., 2002). The global mutation rates measured in several
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reporters by PCR-based technique were estimated to be in the range of 5 × 10−3 –10−5 per nucleotide in mammalian cells expressing AID. Such a level of mutagenesis would generate from thousands to millions of new mutations in the euchromatic part of the genome during each division. When the mutator effect of AID was estimated by the standard genetic method in CHO cells expressing AID, it was found that reversion frequency was increased only 15-fold in comparison to control cells (Martin et al., 2002). AID can also induce mutations when expressed in E. coli (Beale et al., 2004; Petersen-Mahrt et al., 2002). The estimated rates were 10−9 –10−10 per nucleotide in a wild-type strain and an order of magnitude higher in cells unable to excise uracil from DNA. The enhancement of mutagenesis in a uracil-DNA glycosylase-deficient ung1 strain suggests that the deamination of cytosine to uracil in DNA is the cause of mutations induced by AID. Almost all mutations arising upon expression of AID were G-A to A-T transitions, consistent with the DNA deamination model. It has been shown that AID is indeed capable of deaminating single-stranded and supercoiled, double-stranded DNA (Pham et al., 2003, 2005; Shen and Storb, 2004). The DNA sequence context specificity of this reaction resembled the specificity of SHM at G-C pairs (DGYW/WRCH motifs; mutating G-C are underlined, D = A/T/G, H = C/A/T, Y = T/C, R = A/G, W = A/T) (Rogozin and Diaz, 2004) and AID mutation specificity in vitro (GYW/WRC motifs) (Pham et al., 2003, 2005). Therefore, eukaryotic cell-specific components may be necessary for delivery of AID to regions undergoing SHM, but not for AID-induced mutagenesis per se. In a recent paper published in Molecular Immunology, Krause et al. analyzed the RNA and DNA editing activity of AID in yeast using a selectable marker and concluded that the expression of AID in yeast, as opposed to APOBEC1, did not lead to RNA editing and was not sufficient for the generation of point mutations in a highly transcribed gene (Krause et al., 2006). They speculated that cofactors for AID-induced somatic hypermutation, that are present in B cells and a variety of mammalian non-B cells, are missing in yeast (Krause et al., 2006). Here, we present arguments that this conclusion, most likely, is due to lack of power of the approach used by Krause et al. (2006). 2. Materials and methods The collection of mutations in the CAN1 gene induced by the expression of AID in yeast has been described before (Mayorov et al., 2005); and mutations from wild-type and ung1 strains were merged into one spectrum. Spectra of somatic mutations in an artificially synthesized EPS sequence were generated by Storb and co-workers (Kim et al., 1999; Storb et al., 1998). Mutations in the GFP gene induced by the expression of AID in murine fibroblasts were generated by Yoshikawa and co-workers (Yoshikawa et al., 2002). Mutation hotspots were defined using a threshold (Th ) for the number of mutations at a site. The threshold is established by analyzing the frequency distribution derived from a mutation spectrum using the CLUSTERM program (www.itb.cnr.it/webmutation/) (Glazko et al., 1998). Briefly, this program decomposes a mutation spectrum into several homogeneous classes of sites, with each class approximated
by a binomial distribution. Variations in mutation frequencies among sites of the same class are random by definition (mutation probability is the same for all sites within a class), but differences between classes are statistically significant. Each site has a probability P(C) to be assigned to class C. A class with the highest mutation frequency is called the hotspot class. Sites with P(Chotspot ) > 0.95 are be assigned to the hotspot class Chotspot and defined as hotspot sites. This approach ensures that the assignment is statistically significant and robust [see Rogozin et al. (2001) for a detailed discussion of this approach and problems associated with its application]. 3. Results and discussion We studied the genetic consequences of AID expression in yeast using well-established, precise mutation assays available for this organism (Mayorov et al., 2005; Poltoratsky et al., 2004). We found that the expression of the human AID gene was moderately mutagenic in the wild-type strain (mutation rates, ranging from 10−8 to 3 × 10−7 per reporter, were increased up to eight-fold in the presence of AID, depending on the marker) and highly mutagenic (up to 46-fold) in the ung1 uracil-DNA glycosylase-deficient strain (Mayorov et al., 2005). The increase in the mutation rate observed in the yeast wild-type strain is similar to the increase found in CHO cells expressing AID (Martin et al., 2002). The number of detectable positions in the reporters used is not defined, therefore we can only make a safe estimate of the maximum mutation rate per nucleotide. For wild-type strain expressing AID, it is 3.7 × 10−6 . The majority of mutations were at G-C pairs, and DGYW/WRCH motifs (Rogozin and Diaz, 2004) were preferential sites of mutations (Table 1). It was concluded that the intrinsic substrate specificity of AID itself is the primary determinant of mutation hotspots at G-C base pairs during SHM (Mayorov et al., 2005). There are two possible explanations for the discrepancy between the conclusions of these two studies (Mayorov et al., 2005; Krause et al., 2006). The frequency of mutations induced by AID estimated by classical methods is lower in comparison to PCR-based estimates of AID-induced mutation rates in mammalian cells (see above). Most likely, sequencing of 6 × 103 bases by (Krause et al., 2006) failed to detect any mutations in yeast due to the use of wild-type strains (capable of removing Table 1 Mutation hotspots induced by the expression of AID in yeast Position
Sequence
Number of substitutions
DGYW/WRCH variant (D = A/T/G, H = T/A/C)
238 268 299 896 980 1166 1392 1426
GTACAGA AAGCAAA GTGGTAC AAGGTAC TCCGTAT CTGCCGC ATGGTTA ATGCAAG
6 9 5 4 8 4 5 5
WRCA WRCA GGYW GGYW – WRCC GGYW WRCA
The data was taken from (Mayorov et al., 2005), the hotspot threshold Th is four mutations.
I.B. Rogozin, Y.I. Pavlov / Molecular Immunology 43 (2006) 1481–1484 Table 2 DNA context of mutations induced by the expression of AID in murine fibroblasts Start/end of motifs
Substitutions
Hotspots
AGCA 94–97 329–332 560–563 641–643 697–700 715–718
2G → A, 4G → C, 1G → T 1G → A, 1C → T 1G → T, 1C → G 1C → T, 1C → A 3G → A, 1G → T 2G → C, 5C → T
Yes No No No No Yes
TGCA 232–235 296–299 620–623 843–846
2G → A 1G → T, 1G → C, 1C → T 1G → A, 2C → T No mutations
No No No No
AGCT 21–24 107–110 140–143 212–215 248–251 464–467 512–515 623–626 797–800 825–828 831–834
2G → A 2G → C, 3C → T, 1C → A 1G → A, 1G → T, 6G → C, 4C → T 1G → A, 1G → C, 7C → T, 3C → G 5G → A, 1G → T, 1G → C, 2C → T, 1C → G 1G → C, 3C → T 2G → A, 1G → T, 2C → T, 1C → G 3G → A, 1G → C, 1C → T, 1C → G 2G → A, 2G → C 3G → A, 1G → T, 1G → C, 2C → T, 2C → G 2C → T
No No Yes Yes Yes No No No No No
TGCT 301–304 671–674 752–755 930–933
1G → A, 1G → T, 2C → T, 2C → G 5G → A, 2G → T, 4C → T 1G → C No mutations
No Yes No No
The data was taken from (Yoshikawa et al., 2002). Only GC-containing DGYW/WRCH motifs are shown, the hotspot threshold Th is five mutations.
uracil from DNA) and the low resolution of analysis of putative mutations (estimated maximum rate of AID-induced mutations is 3 per 106 nucleotides, see above). When a selective test for mutations was used by Krause and co-workers, the detection limit was apparently low too, less that 1 mutant per 5 × 105 yeast colonies (Krause et al., 2006). In addition, it is possible that the target gene selected by Krause and co-workers had low mutability by DNA deamination (Krause et al., 2006). As correctly pointed out by Krause et al., the sequence of Gal4-VH4 contains several DGYW/WRCH motifs, mutations C → T are detectable in two sequences,
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AGCA and TGCA (Krause et al., 2006). These sequences are mutable in SHM (Rogozin and Diaz, 2004). However, mutable motifs alone are not sufficient for the emergence of hotspots (Rogozin and Pavlov, 2003). We analyzed the distribution of mutations in AGCA, TGCA, AGCT and TGCT sequences induced by expression of AID in murine fibroblasts (Yoshikawa et al., 2002). Only six of the 24 mutable sequences contain mutation hotspots (Table 2). The distribution of mutation hotspot across the GFP gene did not show any obvious pattern: four hotspots are located in the beginning of the GFP gene and two are located towards the end of the gene, and long regions of the gene contain no hotspots (Table 2). Non-uniformity of the hotspot distribution may be also detected for closely located mutable motifs as illustrated by the distribution of somatic mutations across mutable AGCT sequences in an artificially synthesized EPS sequence inserted into an immunoglobulin gene (Fig. 1) (Kim et al., 1999; Storb et al., 1998). The EPS sequence contains AGCT sequences repeated six times, respectively (PA-PF monomeric units in Fig. 1). The number of mutations at G-C bases within AGCT sites varied from four (the PF monomer) to 21 (the PD monomer). Two significantly different classes of AGCT motifs were revealed by the CLUSTERM program (Glazko et al., 1998). The hotspot class includes PA, PB, PC, and PD sequences (Fig. 1). Another class consists of PE and PF sequences which have significantly lower frequencies of mutations. This result shows that a significant heterogeneity of the mutation rate exists even in monotonously repeated AGCT motifs located as far as 600 nucleotides away from the transcription start (Fig. 1). These two examples show that mutation hotspots are not equivalent to mutable motifs. The emergence of hotspots is a complex process that depends on multiple factors. Thus, some mutable motifs are cold spots in an unfavorable global sequence context (Rogozin and Pavlov, 2003). This might add further explanation for the lack of mutations in two DGYW/WRCH motifs observed by Krause and co-workers (Krause et al., 2006). Another possible explanation for the absence of mutations in samples analyzed by Krause and co-workers may be the use of plasmids while other authors studied mutations in chromosomal genes. This explanation is less likely because the detection of recombination events by Krause and co-workers (Krause et al., 2006) suggests that AID in these experiments deaminated the plasmid DNA; it has been demonstrated previously that the expression of AID leads to the induction of mitotic intragenic recombination between chromosomal alleles in uracil-DNA glycosylase-proficient yeast (Poltoratsky et al., 2004).
Fig. 1. Somatic hypermutation spectrum in an artificially synthesized EPS sequence (Kim et al., 1999; Storb et al., 1998). The AGCT mutable motifs are underlined. Position numbers refer to the transcription start site.
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The results of Mayorov et al. (2005) are consistent with the hypothesis that AID-mediated deamination of DNA is a major cause of mutations at G-C base pairs in immunoglobulin genes during SHM. A general pattern of mutations and frequencies of AID-induced substitutions in yeast is similar to the targeting of somatic mutation to the DGYW/WRCH mutable motifs, which are highly specific for SHM in mammals (Rogozin and Diaz, 2004). This indicates that the intrinsic substrate specificity of AID itself is the primary determinant of mutation hotspots at GC base pairs during SHM. Quantitative aspects of the mutability of those genomes exposed to AID vary from species to species and, in multicellular organisms, depend on cell types. Discrepancies among estimates of mutability in the same organisms may be explained by the difference in the genetic background and experimental approaches employed for the analysis of mutagenesis. References Beale, R.C., Petersen-Mahrt, S.K., Watt, I.N., Harris, R.S., Rada, C., Neuberger, M.S., 2004. Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo. J. Mol. Biol. 337, 585–596. Glazko, G.V., Milanesi, L., Rogozin, I.B., 1998. The subclass approach for mutational spectrum analysis: application of the SEM algorithm. J. Theor. Biol. 192, 475–487. Honjo, T., Muramatsu, M., Fagarasan, S., 2004. AID: how does it aid antibody diversity? Immunity 20, 659–668. Honjo, T., Nakai, S., Nishida, Y., Kataoka, T., Yamawaki-Kataoka, Y., Takahashi, N., Obata, M., Shimizu, A., Yaoita, Y., Nikaido, T., Ishida, N., 1981. Rearrangements of immunoglobulin genes during differentiation and evolution. Immunol. Rev. 59, 33–67. Kim, N., Bozek, G., Lo, J.C., Storb, U., 1999. Different mismatch repair deficiencies all have the same effects on somatic hypermutation: intact primary mechanism accompanied by secondary modifications. J. Exp. Med. 190, 21–30. Kinoshita, K., Honjo, T., 2001. Linking class-switch recombination with somatic hypermutation. Nat. Rev. Mol. Cell. Biol. 2, 493–503. Krause, K., Marcu, K.B., Greeve, J., 2006. The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system. Mol. Immunol. 43, 295–307.
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