DNA damage-induced ubiquitylation of RFC2 subunit of replication factor C complex

Junya Tomida, Yuji Masuda, Hidekazu Hiroaki, Tomoko Ishikawa, Ihnyoung Song, Toshiki Tsurimoto, Satoshi Tateishi, Tadahiro Shiomi, Yasuhiro Kamei, Jinhyeong Kim, Kenji Kamiya, Cyrus Vaziri, Haruo Ohmori, Takeshi Todo

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)


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 H2O2. 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.

Original languageEnglish
Pages (from-to)9071-9079
Number of pages9
JournalJournal of Biological Chemistry
Issue number14
Publication statusPublished - Apr 4 2008

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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