En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2-E3 pairs.
Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6-RAD18 (an E2-E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2-UBC13 (a UEV-E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ubn) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.
Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. masuda@riem.nagoya-u.ac.jp
Yuji
Y
Masuda
Miki
M
Suzuki
Hidehiko
H
Kawai
Asami
A
Hishiki
Hiroshi
H
Hashimoto
Chikahide
C
Masutani
Takashi
T
Hishida
Fumio
F
Suzuki
Kenji
K
Kamiya
D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D025821:Polyubiquitin / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
D004268:DNA-Binding Proteins; C100023:HLTF protein, human; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; D014157:Transcription Factors; D025821:Polyubiquitin; C471545:UBE2A protein, human; C471607:UBE2N protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases