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.
Mesh Terms:
Alkylating Agents, Amino Acid Substitution, Cell Line, DNA Damage, DNA Repair, DNA Replication, DNA-Binding Proteins, Humans, Hydrogen Peroxide, Ligases, Multiprotein Complexes, Mutation, Missense, Oxidants, Phosphorylation, Proliferating Cell Nuclear Antigen, Protein Processing, Post-Translational, Replication Protein C, Ubiquitin-Conjugating Enzymes, Ubiquitination
J. Biol. Chem.
Date: Apr. 04, 2008
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