Interaction and colocalization of Rad9/Rad1/Hus1 checkpoint complex with replication protein A in human cells

Wu, Xiaoming; Shell, Steven M.; Zou, Yue

doi:10.1038/sj.onc.1208674

Cited by 103 publications

(101 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Coimmunoprecipitation assays were conducted as previously described (13,15). Four micrograms of rabbit anti-ATR (Oncogene, Cambridge, MA) or anti-XPA (Santa Cruz Biotechnology) were used for each coimmunoprecipitation assay.…”

Section: Methodsmentioning

confidence: 99%

“…The checkpoint signaling cascades, conceptually, consist of three major biochemical components: damage sensors, signal mediators/transducers, and effector molecules. The ataxia-telangiectasia mutated and Rad3-related (ATR) and ataxia-telangiectasia mutated (ATM) checkpoint proteins and the Rad9-Rad1-Hus1/Rad17-Rfc2-5 checkpoint complex have been suggested to be involved in DNA damage recognition and signaling in human cells (2,4,9,(13)(14)(15). ATR and ATM belong to the phosphatidylinositol-3-kinase-related kinase (PIKK) family of proteins.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related–Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

Shell

Yang

et al. 2006

Cancer Research

Self Cite

View full text Add to dashboard Cite

DNA damage triggers complex cellular responses in eukaryotic cells, including initiation of DNA repair and activation of cell cycle checkpoints. In addition to inducing cell cycle arrest, checkpoint also has been suggested to modulate a variety of other cellular processes in response to DNA damage. In this study, we present evidence showing that the cellular function of xeroderma pigmentosum group A (XPA), a major nucleotide excision repair (NER) factor, could be modulated by checkpoint kinase ataxia-telangiectasia mutated and Rad3-related (ATR) in response to UV irradiation. We observed the apparent interaction and colocalization of XPA with ATR in response to UV irradiation. We showed that XPA was a substrate for in vitro phosphorylation by phosphatidylinositol-3-kinase-related kinase family kinases whereas in cells XPA was phosphorylated in an ATR-dependent manner and stimulated by UV irradiation. The Ser196 of XPA was identified as a biologically significant residue to be phosphorylated in vivo. The XPA-deficient cells complemented with XPA-S196A mutant, in which Ser196 was substituted with an alanine, displayed significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Moreover, substitution of Ser196 with aspartic acid for mimicking the phosphorylation of XPA increased the cell survival to UV irradiation. Taken together, our results revealed a potential physical and functional link between NER and the ATRdependent checkpoint pathway in human cells and suggested that the ATR checkpoint pathway could modulate the cellular activity of NER through phosphorylation

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related–Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

Shell

Yang

et al. 2006

Cancer Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…It is possible that mutations in DBD-F lead to disruption of an interaction(s) with repair/recombination proteins or an altered conformation of the RPA complex (perhaps caused by a disruption between the RPA2 N terminus and the RPA1 N terminus) that leads to the observed repair and cell cycle defects. Finally, depletion of RPA1 leads to an inability to load the 9-1-1 complex onto damaged DNA (42). Furthermore, in yeast, rfa1-t11 inhibits recruitment of Rad17 to DNA damage (64), and it has been proposed that this would result in a defect in cell cycle regulation.…”

Section: Mutation Of Key Aromatic Residues Of Rpa1 Reveals the Importmentioning

confidence: 99%

“…This is not surprising as Chk2 is a downstream target of ATM (41). In addition to ATM activation, RPA1 is necessary for association of the Rad9-Rad1-Hus1 (9-1-1) complex with damaged DNA (42).…”

mentioning

confidence: 99%

Cellular Functions of Human RPA1

Haring

Mason

Binz³

et al. 2008

Journal of Biological Chemistry

100

View full text Add to dashboard Cite

In eukaryotes, the single strand DNA (ssDNA)-binding protein, replication protein A (RPA), is essential for DNA replication, repair, and recombination. RPA is composed of the following three subunits: RPA1, RPA2, and RPA3. The RPA1 subunit contains four structurally related domains and is responsible for high affinity ssDNA binding. This study uses a depletion/replacement strategy in human cells to reveal the contributions of each domain to RPA cellular functions. Mutations that substantially decrease ssDNA binding activity do not necessarily disrupt cellular RPA function. Conversely, mutations that only slightly affect ssDNA binding can dramatically affect cellular function. The N terminus of RPA1 is not necessary for DNA replication in the cell; however, this region is important for the cellular response to DNA damage. Highly conserved aromatic residues in the high affinity ssDNA-binding domains are essential for DNA repair and cell cycle progression. Our findings suggest that as long as a threshold of RPA-ssDNA binding activity is met, DNA replication can occur and that an RPA activity separate from ssDNA binding is essential for function in DNA repair.Cell survival and proliferation depend on the efficient maintenance of genetic information. Human cells must accurately replicate billions of base pairs of DNA and identify and repair a wide variety of DNA lesions. One of the proteins required for the maintenance of genomic integrity is replication protein A (RPA). 3 RPA is a heterotrimeric single strand DNA (ssDNA)-binding protein, composed of 70-, 32-, and 14-kDa subunits, commonly referred to as RPA1, RPA2, and RPA3, respectively (1-3). Homologs of the three RPA subunits have been found in all eukaryotes examined (1, 4). The major biochemical activity of RPA is ssDNA binding; RPA can bind ssDNA with subnanomolar affinity (5). The ability of RPA to bind ssDNA is not sequence-specific; however, RPA has a higher affinity for pyrimidine-rich and telomeric ssDNA (6 -8).Replication protein A was originally isolated as a factor essential for in vitro DNA replication of simian virus 40 (SV40) (9 -11), and it has since been shown to be essential for a number of other DNA metabolic processes, including chromosomal DNA replication, repair, and recombination (1-3). RPA also has a role in maintenance of telomeres (7, 12) and in regulation of the cell cycle (13,14). The common feature of all of these processes is that each has ssDNA intermediates that must be recognized and processed appropriately. RPA interacts with ssDNA in the cell and with a number of proteins involved in these processes (3,15).Each of the RPA subunits has at least one domain containing an oligonucleotide/oligosaccharide-binding (OB) fold. This fold is found in many ssDNA-and sugar-binding proteins (16). The OB-folds in RPA are commonly referred to as DNA-binding domains (DBDs). RPA1 contains four OB-folds, and RPA2 and RPA3 have one OB-fold each. Although each OB-fold is structurally similar, the majority of the ssDNA binding occurs through two OB-fo...

show abstract

“…The PML nuclear bodies have been proposed as a scaffold structure for many DDR and repair proteins (43). Upon DNA damage, these proteins such as ATR and RPA undergo dynamic translocation from PML NBs to nuclear foci that represent sites of DNA damage and repair.…”

Section: Rfwd3 Localizes To Sites Of Dna Damage-previouslymentioning

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

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Interaction and colocalization of Rad9/Rad1/Hus1 checkpoint complex with replication protein A in human cells

Cited by 103 publications

References 49 publications

Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related–Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related–Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

Cellular Functions of Human RPA1

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

Contact Info

Product

Resources

About