Incorporation of dUTP does not mediate mutation of A:T base pairs in Ig genes in vivo

Nucleic Acids Res. 2010 Dec;38(22):8120-30. doi: 10.1093/nar/gkq682. Epub 2010 Aug 12.

Abstract

Activation-induced cytidine deaminase (AID) protein initiates Ig gene mutation by deaminating cytosines, converting them into uracils. Excision of AID-induced uracils by uracil-N-glycosylase is responsible for most transversion mutations at G:C base pairs. On the other hand, processing of AID-induced G:U mismatches by mismatch repair factors is responsible for most mutation at Ig A:T base pairs. Why mismatch processing should be error prone is unknown. One theory proposes that long patch excision in G1-phase leads to dUTP-incorporation opposite adenines as a result of the higher G1-phase ratio of nuclear dUTP to dTTP. Subsequent base excision at the A:U base pairs produced could then create non-instructional templates leading to permanent mutations at A:T base pairs (1). This compelling theory has remained untested. We have developed a method to rapidly modify DNA repair pathways in mutating mouse B cells in vivo by transducing Ig knock-in splenic mouse B cells with GFP-tagged retroviruses, then adoptively transferring GFP(+) cells, along with appropriate antigen, into primed congenic hosts. We have used this method to show that dUTP-incorporation is unlikely to be the cause of AID-induced mutation of A:T base pairs, and instead propose that A:T mutations might arise as an indirect consequence of nucleotide paucity during AID-induced DNA repair.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / chemistry*
  • Animals
  • Base Pairing
  • Deoxyuracil Nucleotides / metabolism*
  • Gene Expression
  • Genes, Immunoglobulin*
  • Germinal Center / metabolism
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Mutation*
  • Pyrophosphatases / metabolism
  • Retroviridae / genetics
  • Retroviridae / metabolism
  • Thymine / chemistry*

Substances

  • Deoxyuracil Nucleotides
  • deoxyuridine triphosphate
  • Pyrophosphatases
  • dUTP pyrophosphatase
  • Adenine
  • Thymine