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

J Biol Chem. 2008 Apr 4;283(14):9071-9. doi: 10.1074/jbc.M709835200. Epub 2008 Feb 1.

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 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylating Agents / pharmacology
  • Amino Acid Substitution
  • Cell Line
  • DNA Damage / physiology*
  • DNA Repair / drug effects
  • DNA Repair / physiology
  • DNA Replication / drug effects
  • DNA Replication / physiology
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Humans
  • Hydrogen Peroxide / pharmacology
  • Ligases / genetics
  • Ligases / metabolism
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism*
  • Mutation, Missense
  • Oxidants / pharmacology
  • Phosphorylation / drug effects
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / metabolism
  • Protein Processing, Post-Translational / drug effects
  • Protein Processing, Post-Translational / physiology*
  • Replication Protein C / genetics
  • Replication Protein C / metabolism*
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Conjugating Enzymes / metabolism
  • Ubiquitin-Protein Ligases
  • Ubiquitination / drug effects
  • Ubiquitination / physiology*

Substances

  • Alkylating Agents
  • DNA-Binding Proteins
  • Multiprotein Complexes
  • Oxidants
  • Proliferating Cell Nuclear Antigen
  • RAD18 protein, human
  • RFC1 protein, human
  • Hydrogen Peroxide
  • UBE2N protein, human
  • UBE2V2 protein, human
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligases
  • Replication Protein C
  • Ligases