Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations - PubMed (original) (raw)
Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations
Anjum Sohail et al. Nucleic Acids Res. 2003.
Abstract
Activation-induced cytidine deaminase (AID) is required for the maturation of antibodies in higher vertebrates, where it promotes somatic hypermutation (SHM), class switch recombination and gene conversion. While it is known that SHM requires high levels of transcription of the target genes, it is unclear whether this is because AID targets transcribed genes. We show here that the human AID promotes C to T mutations in Escherichia coli which are stimulated by transcription. The mutations are strand-biased and occur preferentially in the non-transcribed strand of the target gene. Human AID purified from E.coli is active without prior treatment with a ribonuclease and deaminates cytosines in plasmid DNA in vitro. Further, the action of this enzyme is greatly stimulated by the transcription of the target gene in a strand-dependent fashion. These results confirm the prediction that AID may act directly on DNA and show that it can act on transcribing DNA in the absence of specialized DNA structures such as R-loops. It suggests that AID may be recruited to variable genes through transcription without the assistance of other proteins and that the strand bias in SHM may be caused by the preference of AID for the non-transcribed strand.
Figures
Figure 1
Deamination of cytosines in a DNA bubble by AID. (A) The sequence of the duplex used as substrate for the AID reaction is shown. The bottom strand is labeled at the 5′ end with 32P. The cytosine that is the target of AID is indicated by a solid arrow. (B and C) 32P-labeled substrate was incubated with the reagents indicated above each lane and then heated in the presence of NaOH. The buffer contained 50 mM Tris–HCl (B) or 20 mM Tris–HCl (C). Φ is 1,10-phenanthroline.
Figure 2
Structures of plasmids. (A) The relevant genetic elements in the three plasmids are shown. All the plasmids confer resistance to carbenicillin and contain the lacI Q gene (not shown). The product of this gene represses transcription from the promoter UP-tac, but not Pkan. The position of the C·G pair in kanS-94D that is the target for AID action is shown. To emphasize that kanS-94D is inverted in pUP27 compared to pUP21, the relevant text has been inverted. (B) pAB7 and pAB8 are respectively similar to pUP21 and pUP27 except the UP-tac promoter in the latter plasmids is replaced with a T7 RNA polymerase promoter.
Figure 3
The effect of transcription on cytosine deaminations caused by AID in vitro. Plasmid pAB7 (A) or pAB8 (B) DNA was transcribed with T7 RNA polymerase as described in the Materials and Methods. The revertant frequency is (total no. of KanR colonies/total no. of CarbR colonies).
References
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