title: RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.
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 WD repeat 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.
authors.affiliation: Center for Molecular Discovery, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Shangfeng
authors.initials: S
authors.lastname: Liu
authors.forename: Jessica
authors.initials: J
authors.lastname: Chu
authors.forename: Nur
authors.initials: N
authors.lastname: Yucer
authors.forename: Mei
authors.initials: M
authors.lastname: Leng
authors.forename: Shih-Ya
authors.initials: SY
authors.lastname: Wang
authors.forename: Benjamin P C
authors.initials: BP
authors.lastname: Chen
authors.forename: Walter N
authors.initials: WN
authors.lastname: Hittelman
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
mesh_terms: D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D016195:G2 Phase / Q000235:genetics; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D000071156:Promyelocytic Leukemia Protein; D011485:Protein Binding; D021381:Protein Transport; D051135:Rad51 Recombinase / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism*; D016196:S Phase / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: C468783:H2AX protein, human; D006657:Histones; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D051716:Replication Protein A; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; C549038:RFWD3 protein, human; D044767:Ubiquitin-Protein Ligases; D051135:Rad51 Recombinase; C516794:RPA2 protein, human