Deoxyuridine is generated preferentially in the nontranscribed strand of DNA from cells expressing activation-induced cytidine deaminase.
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.
Martomo
Stella A
SA
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Fu
Dongtao
D
Yang
William W
WW
Joshi
Nikhil S
NS
Gearhart
Patricia J
PJ
D001483:Base Sequence; D015139:Blotting, Southern; D003562:Cytidine / Q000378:metabolism; D003564:Cytidine Deaminase; D043525:Cytosine Deaminase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004260:DNA Repair / Q000235:genetics; D003641:Deamination; D003857:Deoxyuridine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D027041:Somatic Hypermutation, Immunoglobulin; D014158:Transcription, Genetic; D014498:Uracil / Q000378:metabolism; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D011994:Recombinant Proteins; D014498:Uracil; D003562:Cytidine; D004247:DNA; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D043525:Cytosine Deaminase; D003564:Cytidine Deaminase; D003857:Deoxyuridine