DNA Damage Induced Hyperphosphorylation of Replication Protein A. 1. Identification of Novel Sites of Phosphorylation in Response to DNA Damage

Nuss, Jonathan E.; Patrick, Steve M.; Oakley, Gregory G.; Alter, Gerald M.; Robison, Jacob G.; Dixon, Kathleen; Turchi, John J.

doi:10.1021/bi0480584

Cited by 72 publications

(70 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The middle subunit of RPA, RPA32, is phosphorylated primarily at two sites, serine 23 and serine 29, during the normal cell cycle in the G 1 /S transition, G 2 and M-phase, followed by dephosphorylation at the end of M-phase (Din et al, 1990). DNA-damaging agents known to stall replication, including UV, inhibitors of topoisomerase I and II and hydroxyurea (HU), induce hyperphosphorylation of RPA at additional serines and threonines on the N-terminus of RPA32, including serines 4, 8 and 33 and threonine 21 (Block et al, 2004;Nuss et al, 2005;Zernik-Kobak et al, 1997). RPA phosphorylation is thought to modulate protein-protein interactions as well as its ability to bind dsDNA (Abramova et al, 1997;Binz et al, 2003;Liu et al, 2005;Patrick et al, 2005;Yoo et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse

Manthey

Opiyo

Glanzer

et al. 2007

Journal of Cell Science

View full text Add to dashboard Cite

Post-translational phosphorylation of proteins provides a mechanism for cells to switch on or off many diverse processes, including responses to replication stress. Replication-stress-induced phosphorylation enables the rapid activation of numerous proteins involved in DNA replication, DNA repair and cell cycle checkpoints, including replication protein A (RPA). Here, we report that hydroxyurea (HU)-induced RPA phosphorylation requires both NBS1 (NBN) and NBS1 phosphorylation. Transfection of both phosphospecific and nonphosphospecific anti-NBS1 antibodies blocked hyperphosphorylation of RPA in HeLa cells. Nijmegen breakage syndrome (NBS) cells stably transfected with an empty vector or with S343A-NBS1 or S278A/S343A phospho-mutants were unable to hyperphosphorylate RPA in DNA-damage-associated foci following HU treatment. The stable transfection of fully functional NBS1 in NBS cells restored RPA hyperphosphorylation. Retention of ATR on chromatin in both NBS cells and in NBS cells expressing S278A/S343A NBS1 mutants decreased after DNA damage, suggesting that ATR is the kinase responsible for RPA phosphorylation. The importance of RPA hyperphosphorylation is demonstrated by the ability of cells expressing a phospho-mutant form of RPA32 (RPA2) to suppress and delay HU-induced apoptosis. Our findings suggest that RPA hyperphosphorylation requires NBS1 and is important for the cellular response to DNA damage.

show abstract

Section: Introductionmentioning

confidence: 99%

NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse

Manthey

Opiyo

Glanzer

et al. 2007

Journal of Cell Science

View full text Add to dashboard Cite

show abstract

“…The phosphorylation of the 32-kDa subunit of RPA2 is well characterized in these processes. At least 10 phosphorylation sites (Ser-4, Ser-8, Ser-10, Ser-11, Ser-12, Thr-21, Ser-23, Ser-29, Ser-33, and Thr-98) and 4 kinases (ATM, ATR, DNA-PKcs, Cdk1, and Cdk2) have been suggested (25)(26)(27)(28)(29)(30)(31)(32). It is shown that DNA damage-induced RPA hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair after replication block but is not essential for IR and I-Sce-I endonuclease-stimulated HR (28).…”

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

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

“…Of the ϳ70-kDa (RPA1), 30-kDa (RPA2), and 14-kDa (RPA3) subunits, human RPA is subject to extensive phosphorylation on RPA2 (2) and at one RPA1 site (15). The N-terminal 33 residues of RPA2 undergo both cell cycle-and stress-dependent phosphorylation on approximately nine sites (Fig.…”

mentioning

confidence: 99%

Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA Repair

Anantha

Vassin

Borowiec

2007

Journal of Biological Chemistry

150

211

View full text Add to dashboard Cite

The activity of human replication protein A (RPA) in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are modified in vivo and in response to genotoxic stress. Using a novel antibody against phospho-Ser 29 , a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in nonstressed cells. Robust phosphorylation of Ser 29 was also seen in interphase cells following treatment with the DNA-damaging agent camptothecin, a rare example of stress stimulating the modification of a repair factor by cyclin-Cdk. RPA2 phosphorylation is regulated both in cis and trans. Cis-phosphorylation follows a preferred pathway. (That is, the initial modification of Ser 33 by ATR stimulates subsequent phosphorylation of Cdk sites Ser 23 and Ser 29 ). These events then facilitate modification of Thr 21 and extreme N-terminal sites Ser 4 and Ser 8 , probably by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans. Cells in which endogenous RPA2 was "replaced" with a double S23A/S29A-RPA2 mutant were seen to have an abnormal cell cycle distribution both in normal and in stressed cells. Such cells also showed aberrant DNA damage-dependent RPA foci and had persistent staining of ␥H2AX following DNA damage. Our data indicate that RPA phosphorylation facilitates chromosomal DNA repair. We postulate that the RPA phosphorylation pattern provides a means to regulate the DNA repair pathway utilized.Replication protein A (RPA) 2 is a heterotrimeric singlestranded DNA-binding factor that is critical for the "three Rs" of eukaryotic DNA enzymology: DNA replication, DNA recombination, and DNA repair (1, 2). For DNA replication, the study of cellular and viral model systems demonstrates that RPA is needed both for origin denaturation and replication elongation, in the latter case to facilitate the switch from DNA polymerase ␣ to DNA polymerase ␦ during Okazaki fragment synthesis (3). RPA acts in homologous recombination (HR) to stimulate DNA annealing using physical interactions with Rad52 (4 -7) and in HR-mediated DNA repair, probably employing specific interactions with BRCA2 (8, 9). RPA is a required factor in both the nucleotide excision (10, 11) and mismatch repair pathways (12, 13) and in somatic hypermutation (14). Because of these many roles, it is of significant interest to understand the mechanisms that regulate RPA activity.Of the ϳ70-kDa (RPA1), 30-kDa (RPA2), and 14-kDa (RPA3) subunits, human RPA is subject to extensive phosphorylation on RPA2 (2) and at one RPA1 site (15). The N-terminal 33 residues of RPA2 undergo both cell cycle-and stress-dependent phosphorylation on approximately nine sites (Fig. 1A), which are thought to exist in an unstructured conformation (16,17). Ser 23 and Ser 29 are constitutively modified during mitosis by cyclin B-Cdk1 (18, 19) and have been suggested to be partially modified beginni...

show abstract

DNA Damage Induced Hyperphosphorylation of Replication Protein A. 1. Identification of Novel Sites of Phosphorylation in Response to DNA Damage

Cited by 72 publications

References 30 publications

NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse

NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse

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

Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA Repair

Contact Info

Product

Resources

About