Analysis of the Human Replication Protein A:Rad52 Complex: Evidence for Crosstalk Between RPA32, RPA70, Rad52 and DNA

Jackson, Doba; Dhar, Kajari; Wahl, James K.; Wold, Marc S.; Borgstahl, Gloria E. O.

doi:10.1016/s0022-2836(02)00541-7

Cited by 66 publications

(69 citation statements)

References 53 publications

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“…aRPA interacted with Rad52 at a level similar to RPA, even though Rad52-RPA interactions are mediated through both RPA1 and RPA2 ( Fig. 2A) (32). In contrast to Rad52, aRPA exhibited a decreased interaction with Rad51 when compared with RPA ( Fig.…”

Section: Rpa4 Mrna Is Found In Normal Humanmentioning

confidence: 89%

“…The purification of the core nucleotide excision repair factors XPA, XPC, XPF-ERCC1, XPG, and TFIIH was reported earlier (38). Recombinant DNA Rad51 and Rad52 were purified as described previously (32,39).…”

Section: Methodsmentioning

confidence: 99%

“…A 70-mer single-stranded DNA (B20T2) was 3Ј end labeled using 3Ј-[␣- 32 P]dATP and terminal transfer-ase (New England Biolabs). This labeled oligomer was purified from a denaturing polyacrylamide gel and annealed to the 70-mer single-stranded DNA (B20C2), which contains 25 nucleotides (nts) of a complementary sequence flanking a central region of 20 nt of unpaired base pairs.…”

Section: Methodsmentioning

confidence: 99%

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

Kemp

Mason

Carreira

et al. 2010

Journal of Biological Chemistry

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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...

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Section: Rpa4 Mrna Is Found In Normal Humanmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Kemp

Mason

Carreira

et al. 2010

Journal of Biological Chemistry

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“…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

Journal of Biological Chemistry

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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...

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“…hRad52 is a key recombination mediator protein that plays a significant role in the initial steps of DNA double strand break repair via specific interactions with proteins of homologous recombination machinery, particularly with Rad51 [1][2][3][4][5][6][7] and Replication protein A [8,9]. Genetic studies point towards roles of Rad52 in Rad51recombinase -dependent [3] and -independent recombination pathways [10,11].…”

Section: Introductionmentioning

confidence: 99%

Salt Modulates Oligomerization Properties of hRad51 and hRad52 Proteins

Balakrishnan¹,

Krishnan²,

Rao³

2009

TOBIOJ

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Human Rad52 (hRad52) and Rad51 (hRad51) proteins are important components of homologous recombination machinery involved in DNA double strand break repair. hRad52 subunits oligomerize to form rings, which are further believed to stack one over another giving rise to higher order structures. Such structures bind the ends of duplex DNA to bring about DNA end joining. hRad51 exists in the native state as oligomeric rings and monomerizes to interact with the DNA. In our current study, we report disruption and solubilization of hRad52 aggregates and higher order aggregation of hRad51 molecules at high salt (KCl) concentration. Computational analysis of the crystal structure available for N-terminal 212 amino acids of hRad52 protein reveal a dense unique distribution of salt bridges, not only between adjacent but also between penultimate subunit neighbors which perhaps contribute to stabilization of hRad52 oligomeric rings. Our results suggest that disruption of inter-subunit salt bridges and thereby perturbation of interaction between individual monomers as the underlying mechanism for salt mediated monomerization of hRad52 protein. The crystal structure of Rad51 on the other hand lacks such dense salt-bridge connectivity suggesting that salt-mediated monomerization is a feature of proteins with dense salt-bridge networks. Salt brings together the hydrophobic surface residues of hRad51 in a process termed as "salting out" resulting in aggregation of hRad51 molecules. Given the functional relevance of oligomeric hRad52 and monomeric hRad51 in homologous recombination mediated repair, our findings imply that salt regulates the oligomerization status of these repair proteins, and thereby, their functions respectively.

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Analysis of the Human Replication Protein A:Rad52 Complex: Evidence for Crosstalk Between RPA32, RPA70, Rad52 and DNA

Cited by 66 publications

References 53 publications

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

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

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

Salt Modulates Oligomerization Properties of hRad51 and hRad52 Proteins

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