Recombinogenic Phenotype of Human Activation-Induced Cytosine Deaminase

Poltoratsky, Vladimir; Wilson, Samuel H.; Kunkel, Thomas A.; Pavlov, Youri I.

doi:10.4049/jimmunol.172.7.4308

Cited by 48 publications

(56 citation statements)

References 55 publications

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“…This is facilitated by its small size (24 kDa) and bona fide nucleocytoplasmic shuttling signals (52). These properties may also contribute to its ability to mutate the yeast CAN1 gene, as reported recently by Poltoratsky et al (53). In contrast, to accomplish retroelement restriction APOBEC3F and -G may never have to leave the cytoplasm, where they can access assembling retroelements (exogenous or endogenous).…”

Section: Discussionmentioning

confidence: 88%

APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast

Schumacher

Nissley

Harris

2005

Proc. Natl. Acad. Sci. U.S.A.

137

111

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Human cells harbor a variety of factors that function to block the proliferation of foreign nucleic acid. The APOBEC3G enzyme inhibits the replication of retroviruses by deaminating nascent retroviral cDNA cytosines to uracils, lesions that can result in lethal levels of hypermutation. Here, we demonstrate that APOBEC3G is capable of deaminating genomic cytosines in Saccharomyces cerevisiae. APOBEC3G expression caused a 20-fold increase in frequency of mutation to canavanine-resistance, which was further elevated in a uracil DNA glycosylase-deficient background. All APOBEC3G-induced base substitution mutations mapped to the nuclear CAN1 gene and were exclusively C͞G 3 T͞A transition mutations within a 5 -CC consensus. The APOBEC3G preferred sites were found on both strands of the DNA duplex, but were otherwise located in hotspots nearly identical to those found previously in retroviral cDNA. This unique genetic system further enabled us to show that expression of APOBEC3G or its homolog APOBEC3F was able to inhibit the mobility of the retrotransposon Ty1 by a mechanism that involves the deamination of cDNA cytosines. Thus, these data expand the range of likely APOBEC3 targets to include nuclear DNA and endogenous retroelements, which have pathological and physiological implications, respectively. We postulate that the APOBEC3-dependent innate cellular defense constitutes a tightly regulated arm of a conserved mobile nucleic acid restriction mechanism that is poised to limit internal as well as external assaults.APOBEC3F ͉ endogenous retrotransposon Ty1 ͉ hypermutation ͉ cytodeamination ͉ Saccharomyces cerevisiae

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Section: Discussionmentioning

confidence: 88%

APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast

Schumacher

Nissley

Harris

2005

Proc. Natl. Acad. Sci. U.S.A.

137

111

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“…In vitro and in vivo experiments in E. coli have shown the transcription dependency of AID deamination and hypermutation, respectively (5)(6)(7)(8)12). A hyperrecombination and hypermutation effect of AID was also observed in yeast (28), but whether this was transcriptiondependent was not determined. In this work we show that hypermutation and hyperrecombination caused by AID in yeast are also transcription-dependent, confirming the hypothesis that the action of AID requires transcription of the target sequence (4,6,12).…”

Section: Discussionmentioning

confidence: 99%

“…To test this possibility, we confirmed that AID can induce hypermutation and hyperrecombination at low levels in yeast cells (28,29) and demonstrated additionally that this instability occurs in a transcription-dependent manner. Using yeast THO mutants as a way to generate suboptimal mRNP formation, we found that both hypermutation and hyperrecombination were strongly and synergistically increased in a transcription-dependent manner, with mutations primarily occurring in the NT strand.…”

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confidence: 90%

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Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex

Gómez-González

Aguilera

2007

Proc. Natl. Acad. Sci. U.S.A.

102

111

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Activation-induced cytidine deaminase (AID) is a B cell enzyme essential for Ig somatic hypermutation and class switch recombination. AID acts on ssDNA, and switch regions of Ig genes, a target of AID, form R-loops that contain ssDNA. Nevertheless, how AID action is specifically targeted to particular DNA sequences is not clear. Because mutations altering cotranscriptional messenger ribonucleoprotein (mRNP) formation such as those in THO/TREX in yeast promote R-loops, we investigated whether the cotranscriptional assembly of mRNPs could affect AID targeting. Here we show that AID action is transcription-dependent in yeast and that strong and transcription-dependent hypermutation and hyperrecombination are induced by AID if cells are deprived of THO. In these strains AID-induced mutations occurred preferentially at WRC motifs in the nontranscribed DNA strand. We propose that a suboptimal cotranscriptional mRNP assembly at particular DNA regions could play an important role in Ig diversification and genome dynamics.hyperrecombination ͉ hypermutation ͉ messenger ribonucleoprotein biogenesis ͉ R-loops A ctivation-induced cytidine deaminase (AID) is a specific B cell enzyme believed to be responsible for the initiation of somatic hypermutation (SHM) and class switch recombination (CSR) during B cell differentiation (1-3). Many studies have shown that AID acts directly on DNA (4), the natural target for AID action in the variable and switch (S) regions for SHM and CSR, respectively. The specific mechanisms of AID function are still unclear, but evidence suggests that the preferential target of AID may be ssDNA (5, 6). Thus, in vitro experiments have shown that AID deaminates ssDNA and dsDNA that is transcribed (5-7), and transcription has been shown to be required for both SHM and CSR in vivo (8,9).Studies have shown a strand preference for the action of AID during in vitro transcription with AID-induced mutations detected preferentially in the nontranscribed (NT) strand (6,(10)(11)(12). Analysis of the products of SHM in B cells, however, reveals that both DNA strands are mutated (13). Other in vitro studies have shown that AID can deaminate both strands within regions of supercoiled DNA (14) and that the ssDNA-binding replication protein A is required for deamination of SHM targets (15). All of these findings are consistent with the idea that transient formation of ssDNA during transcription facilitates AID action.Along the same line, formation of R-loops during transcription of the S regions has been proposed (16). Such R-loops would possibly provide AID with ssDNA substrates at its target region because there the transcribed (T) strand hybridizes with the nascent RNA and the NT strand is present as ssDNA. Because of its high G content, the NT DNA strand at S regions could be stabilized by the formation of parallel four-stranded G quartets (17, 18). Indeed, AID appears to bind specifically to G-loops within transcribed S regions (19) and can deaminate the displaced strand of a transcription-induced R-loop in vitro ...

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“…In contrast, copious genetic and biochemical evidence supports the DNA deamination theory that AID deaminates cytidine to uridine in DNA. Regarding genetics, overexpression of AID in Escherichia coli (4), yeast (5), cultured cell lines (6 -10), and mice (11) produced mostly C to T transitions in DNA, which likely resulted from a DNA polymerase replicating deoxyuridine (dU). Nonetheless, overexpression of APOBEC1 in E. coli also produced C to T transitions (12), suggesting that this assay is not specific for determining enzyme activity in mammalian cells.…”

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confidence: 99%

Deoxyuridine Is Generated Preferentially in the Nontranscribed Strand of DNA from Cells Expressing Activation-Induced Cytidine Deaminase

Martomo

Yang

et al. 2005

The Journal of Immunology

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Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and class switch recombination of Ig genes in B cells. Although AID has been shown to deaminate deoxycytidine to deoxyuridine in DNA in vitro, there is no physical evidence for increased uracils in DNA from cells expressing AID in vivo. We used several techniques to detect uracil bases in a gene that was actively transcribed in Escherichia coli cells expressing AID. Plasmid DNA containing the gene was digested with uracil-DNA glycosylase to remove uracil, and apurinic/apryimidinic endonuclease to nick the abasic site. The nicked DNA was first analyzed using alkaline gel electrophoresis, in which there was a 2-fold increase in the linear form of the plasmid after AID induction compared with plasmid from noninduced bacteria. Second, using a quantitative denaturing Southern blot technique, the gene was predominantly nicked in the nontranscribed strand compared with the transcribed strand. Third, using ligation-mediated PCR, the nicks were mapped on the nontranscribed strand and were located primarily at cytosine bases. These data present direct evidence for the presence of uracils in DNA from cells that are induced to express AID, and they are preferentially generated at cytosines in the nontranscribed strand during transcription.

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Recombinogenic Phenotype of Human Activation-Induced Cytosine Deaminase

Cited by 48 publications

References 55 publications

APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast

APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast

Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex

Deoxyuridine Is Generated Preferentially in the Nontranscribed Strand of DNA from Cells Expressing Activation-Induced Cytidine Deaminase

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