UBE2V2 (MMS2) is not required for effective immunoglobulin gene conversion or DNA damage tolerance in DT40.
The RAD6/RAD18 heterodimer promotes translesion synthesis via the monoubiquitination of the DNA sliding clamp, PCNA. In S. cerevisiae, a second complex, UBC13/MMS2/RAD5, can extend this single ubiquitin with a non-canonical lysine 63-linked chain. This polyubiquitination step is required for an error-free mode of bypass, possibly template switching by the stalled replication complex. Evidence of a role for the human homologue of MMS2, UBE2V2, in such a process has been inferred from the abrogation of ultraviolet light-induced gene conversion following antisense knockdown of the transcript in human fibroblasts. To ask whether a similar mechanism contributes to abasic site-induced immunoglobulin gene conversion, and to ascertain the role of UBE2V2 in the vertebrate DNA damage response we created a ube2v2 mutant of the chicken cell line DT40. Unlike budding yeast mms2, ube2v2 DT40 does not exhibit significant hypersensitivity to DNA damage, nor the elevated sister chromatid exchange seen in vertebrate rad18 mutants suggesting that UBE2V2 plays a minor or redundant role in RAD18 dependent DNA damage tolerance. In addition, both ube2v2 and rad18 DT40 display more or less normal levels of immunoglobulin gene conversion and, despite the important role played by RAD18 in DNA damage induced translesion synthesis, rad18 DT40, unlike rev1 DT40, does not show a defect in non-templated immunoglobulin gene mutation. Together these data suggest that signalling through PCNA ubiquitination is not required for immunoglobulin diversification in DT40.
Simpson
Laura J
LJ
MRC Laboratory of Molecular Biology, Division of Protein & Nucleic Acid Chemistry, Hills Road, Cambridge, CB2 2QH, UK.
Sale
Julian E
JE
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D004249:DNA Damage; D005785:Gene Conversion; D014644:Genetic Variation; D008025:Ligases / Q000378:metabolism*; D009153:Mutagens / Q000633:toxicity; D012854:Sister Chromatid Exchange; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D014466:Ultraviolet Rays
D009153:Mutagens; C114715:UBE2V2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases