Physical Interaction between Replication Protein A and Rad51 Promotes Exchange on Single-stranded DNA

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 ␥H2AX 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.The cellular response to genotoxic stress initiates multiple signal transduction pathways that include transcription regulation, cell cycle arrest, DNA damage repair, and apoptosis (1, 2). Central to DDR 2 are two checkpoint kinases ATM and ATR that phosphorylate many downstream effectors to execute these functions (3, 4). Identification of key players and the characterization of their molecular functions enable a more thorough understanding of the DDR pathways, which are critical for maintaining genomic integrity. Several recent proteomic screens have drastically expanded the landscape of DDR pathways with the identification of hundreds of putative ATM/ATR substrates (5-7). Recently, we investigated the role of a previously uncharacterized protein, RING finger and WD repeat domain 3 (RFWD3, also known as RNF201 and FLJ10520; GenBank TM number 55159) in DDR (5, 8). Originally identified from a proteomic screen for ATM/ATR substrates, RFWD3 was found phosphorylated at several conserved SQ sites in cells that were treated with ionizing radiation (IR) and replication blocking agents. Subsequent biochemical analysis revealed that RFWD3 can form a complex with MDM2 and p53 and is required for the maintenance of high levels of p53 upon DNA damage induction. The RFWD3 protein contains several well characterized functional domains as well as domains of unknown functions. The N-terminal RING finger domain is a conserved E3 ubiquitin (Ub) ligase domain. In vitro r...

Section: Rfwd3 Localizes To Sites Of Dna Damage-previouslymentioning

confidence: 99%

“…RPA is also shown to be directly involved in homology-directed repair. RPA interacts with homologous recombination (HR) repair protein Rad51, Rad52, and BRCA2 (15)(16)(17)(18)(19)(20)(21)(22)(23). Depletion of RPA by siRNA knockdown impairs the recruitment of Rad51 to sites of DNA repair and increases sensitivity to DNA damaging agents (18,24).…”

mentioning

confidence: 99%

RING Finger and WD Repeat Domain 3 (RFWD3) Associates with Replication Protein A (RPA) and Facilitates RPA-mediated DNA Damage Response

Liu

Chu

Yucer

et al. 2011

“…Although the exact role of Mcm10 in replication initiation has yet to be elucidated, it is reasonable to envision Mcm10 as a macromolecular recruiting and/or scaffolding protein because of the fact that Mcm10 contains two domains that can bind to DNA and pol ␣ (29). This follows a common strategy for numerous modular proteins involved in DNA processing; there is a significant kinetic advantage to deconstructing protein interactions into two or more weak binding sites (68). The recruitment of pol ␣ to origins of replication by Mcm10 would be a significant step to signal nascent DNA synthesis and contribute to fork stability (6,12,16,24,26,27,29,71).…”

Section: A Molecular Mechanism For Mcm10mentioning

confidence: 99%

Physical Interactions between Mcm10, DNA, and DNA Polymerase α

Warren

Huang²,

Fanning³

et al. 2009

Self Cite

Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase ␣ (pol ␣), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol ␣. However, the mechanism by which Mcm10 interacts with pol ␣ on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID⅐ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286 -310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol ␣ within the replication fork.

“…2A). Rad51 also interacts with both RPA1 and RPA2 (30,46). To explore domains involved in the altered interactions, we also examined interactions with a mutant form of RPA1 composed solely of two copies of a fragment of the core DNA binding domain, AA-His (containing residues 177-303 of RPA1) (47).…”

Section: Rpa4 Mrna Is Found In Normal Humanmentioning

confidence: 99%

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

Kemp

Mason

Carreira

et al. 2010

Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells. Replication protein A (RPA)3 is the major single-stranded DNA-binding protein in human cells (1-3). It is composed of subunits of 70, 32, and 14 kDa (RPA1, the 70-kDa subunit of RPA; RPA2; and RPA3, the 14-kDa subunit of RPA, respectively) and was originally identified as an essential component for simian virus 40 (SV40) replication (1). RPA has since been shown to be essential for DNA replication, DNA repair, recombination, and coordination of the cellular response to DNA damage.In addition to the three canonical subunits of RPA (RPA1, RPA2, and RPA3), the human genome contains an additional subunit called RPA4 that is 63% similar to RPA2.4 RPA4 was originally identified in a screen for proteins that interact with RPA1 (5). RPA4 is an intronless gene on the X chromosome, and RPA4 homologs with complete coding sequences are only found in primates and horse. 4 Initial analysis indicated that at least some human tissues express RPA4 protein, though its role in these tissues was not determined (5). RPA4 protein can substitute for RPA2 in the RPA complex, forming an alternative RPA complex (aRPA) that has biochemical properties similar to canonical RPA (6). Surprisingly, whereas RPA is essential for DNA synthesis in the SV40 replication system, aRPA failed to substitute for RPA and acted in a dominant-negative fashion to inhibit DNA replication in the presence of canonical RPA (6). In addition, studies of RPA2-depleted HeLa cells expressing RPA4 demonstrated that aRPA is not able to support S-phase progr...