RING Finger and WD Repeat Domain 3 (RFWD3) associates with Replication Protein A (RPA) and facilitates RPA-mediated DNA damage response

Shangfeng Liu, Jessica Chu, Nur Yucer, Mei Leng, Shih Ya Wang, Benjamin P C Chen, Walter N. Hittelman, Yi Wang

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WDrepeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.

Original languageEnglish (US)
Pages (from-to)22314-22322
Number of pages9
JournalJournal of Biological Chemistry
Volume286
Issue number25
DOIs
StatePublished - Jun 24 2011

Fingerprint

Replication Protein A
DNA Damage
DNA
DNA Repair
Repair
Automatic teller machines
RING Finger Domains
WD40 Repeats
Recombinational DNA Repair
Ubiquitin-Protein Ligases
Single-Stranded DNA
Chemical activation
DNA-Binding Proteins
DNA Replication
Genetic Markers
Proteomics
Phosphorylation
Carrier Proteins
Phosphotransferases
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

RING Finger and WD Repeat Domain 3 (RFWD3) associates with Replication Protein A (RPA) and facilitates RPA-mediated DNA damage response. / Liu, Shangfeng; Chu, Jessica; Yucer, Nur; Leng, Mei; Wang, Shih Ya; Chen, Benjamin P C; Hittelman, Walter N.; Wang, Yi.

In: Journal of Biological Chemistry, Vol. 286, No. 25, 24.06.2011, p. 22314-22322.

Research output: Contribution to journalArticle

Liu, Shangfeng ; Chu, Jessica ; Yucer, Nur ; Leng, Mei ; Wang, Shih Ya ; Chen, Benjamin P C ; Hittelman, Walter N. ; Wang, Yi. / RING Finger and WD Repeat Domain 3 (RFWD3) associates with Replication Protein A (RPA) and facilitates RPA-mediated DNA damage response. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 25. pp. 22314-22322.
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