Incorporation of dUTP does not mediate mutation of A:T base pairs in Ig genes in vivo.
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.
Sharbeen
George
G
Centenary Institute, The University of Sydney, Sydney NSW 2006, Australia.
Cook
Adam J L
AJ
Lau
K K Edwin
KK
Raftery
Joanna
J
Yee
Christine W Y
CW
Jolly
Christopher J
CJ
D000225:Adenine / Q000737:chemistry*; D000818:Animals; D020029:Base Pairing; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D018858:Germinal Center / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D011755:Pyrophosphatases / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D013941:Thymine / Q000737:chemistry*
D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D000225:Adenine; D013941:Thymine