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

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22 Citations (Scopus)

Abstract

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
Volume283
Issue number14
DOIs
Publication statusPublished - Apr 4 2008

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Tomida, J., Masuda, Y., Hiroaki, H., Ishikawa, T., Song, I., Tsurimoto, T., Tateishi, S., Shiomi, T., Kamei, Y., Kim, J., Kamiya, K., Vaziri, C., Ohmori, H., & Todo, T. (2008). DNA damage-induced ubiquitylation of RFC2 subunit of replication factor C complex. Journal of Biological Chemistry, 283(14), 9071-9079. https://doi.org/10.1074/jbc.M709835200