hMMS2 serves a redundant role in human PCNA polyubiquitination.
BACKGROUND
In yeast, DNA damage leads to the mono and polyubiquitination of the sliding clamp PCNA. Monoubiquitination of PCNA is controlled by RAD18 (E3 ligase) and RAD6 (E2 conjugating enzyme), while the extension of the monoubiquitinated PCNA into a polyubiquitinated substrate is governed by RAD5, and the heterodimer of UBC13/MMS2. Each modification directs a different branch of the DNA damage tolerance pathway (DDT). While PCNA monoubiquitination leads to error-prone bypass via TLS, biochemical studies have identified MMS2 along with its heteromeric partner UBC13 to govern the error-free repair of DNA lesions by catalyzing the formation of lysine 63-linked polyubiquitin chains (K63-polyUb). Recently, it was shown that PCNA polyubiquitination is conserved in human cells and that this modification is dependent on RAD18, UBC13 and SHPRH. However, the role of hMMS2 in this process was not specifically addressed.


RESULTS
In this report we show that mammalian cells in which MMS2 was reduced by siRNA-mediated knockdown maintains PCNA polyubiquitination while a knockdown of RAD18 or UBC13 abrogates PCNA ubiquitination. Moreover, the additional knockdown of a UEV1A (MMS2 homolog) does not deplete PCNA polyubiquitination. Finally, mouse embryonic stem cells null for MMS2 with or without the additional depletion of mUEV1A continue to polyubiquitinated PCNA with normal kinetics.


CONCLUSIONS
Our results point to a high level of redundancy in the DDT pathway and suggest the existence of another hMMS2 variant (hMMSv) or complex that can compensate for its loss.
Brun
Jan
J
Centre for Cancer Therapeutics, Ottawa Health Research Institute, Ottawa, ON K1H 8L6, Canada. jzbrun@yahoo.com
Chiu
Roland
R
Lockhart
Katherine
K
Xiao
Wei
W
Wouters
Bradly G
BG
Gray
Douglas A
DA
D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000172:deficiency / Q000378:metabolism*; D051379:Mice; D025821:Polyubiquitin / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism / Q000528:radiation effects; D014157:Transcription Factors / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000172:deficiency / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination / Q000528:radiation effects; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; D034741:RNA, Small Interfering; D014157:Transcription Factors; D025821:Polyubiquitin; C471607:UBE2N protein, human; C471624:UBE2V1 protein, human; C114715:UBE2V2 protein, human; C511778:Ube2v2 protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases