DNA damage-induced ubiquitylation of RFC2 subunit of replication factor C complex.
Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.
Tomida
Junya
J
Radiation Biology Center, and Institute for Virus Research, Kyoto University, Kyoto 606-8501, Japan.
Masuda
Yuji
Y
Hiroaki
Hidekazu
H
Ishikawa
Tomoko
T
Song
Ihnyoung
I
Tsurimoto
Toshiki
T
Tateishi
Satoshi
S
Shiomi
Tadahiro
T
Kamei
Yasuhiro
Y
Kim
Jinhyeong
J
Kamiya
Kenji
K
Vaziri
Cyrus
C
Ohmori
Haruo
H
Todo
Takeshi
T
D000477:Alkylating Agents / Q000494:pharmacology; D019943:Amino Acid Substitution; D002460:Cell Line; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D016877:Oxidants / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D051818:Replication Protein C / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology*
D000477:Alkylating Agents; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D016877:Oxidants; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; C081632:RFC1 protein, human; D006861:Hydrogen Peroxide; C471607:UBE2N protein, human; C114715:UBE2V2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D051818:Replication Protein C; D008025:Ligases