RPA2 Research Articles

Using budding yeast to study properties and function of the human Replication Protein A2 N‐terminus in response to specific‐types of DNA damage

BackgroundReplication Protein A (RPA) is a heterotrimeric complex that serves critical roles in eukaryotic genome duplication and maintenance. In human cells, the N‐terminus (NT) of the 32‐kDa subunit of RPA (Rpa2) is hyper‐phosphorylated by multiple kinases in response to DNA damage. We demonstrated previously in the budding yeast Saccharomyces cerevisiae that phosphorylation of the yeast Rpa2 NT does not occur under commonly assayed genotoxic conditions, yet this domain is vital for the damage response. Using a chimeric yeast Rpa2 protein containing the human Rpa2 N‐terminus, we have demonstrated that this human domain is phosphorylated in yeast cells a manner nearly indistinguishable from that in human cells.Study ObjectiveWe wish to determine properties of human Rpa2 NT phosphorylation, including kinases required, sites involved and their interdependence, and what conditions trigger this phosphorylation to identify whether properties of Rpa2 NT phosphorylation are conserved in yeast and human cells.MethodsWe utilized chimeric human‐yeast Rpa2 proteins (with WT and mutant NT), yeast Rpa2 antibodies, and human Rpa2 phospho‐specific antibodies to examine the potential for the human Rpa2 NT to be phosphorylated in response to varying DNA damaging agents.Results and ConclusionsWe determined that the same sites in the human Rpa2 NT are phosphorylated on the chimeric protein in yeast, and that these show similar dependence on DNA damage induction. Unlike that reported in human cells, phosphorylation of one site does not depend on phosphorylation of another site within the domain (i.e., no sequential/primed phosphorylation). We demonstrated that phosphorylation of the human Rpa2 NT in yeast only appears to occur when cells are treated with agents that produce double‐strand breaks (e.g., HO endonuclease, phleomycin, camptothecin) but not when replication is inhibited by hydroxyurea. Finally, we have shown that Mec1 (yeast ATR), but surprisingly not Tel1 (yeast ATM), is necessary to phosphorylate T21 and S33 (both containing TQ or SQ) on the human Rpa2 NT. This is in contrast to human cells where ATR has only been reported to phosphorylate S33, not T21. We conclude that yeast provides a simple system to identify kinases involved, conditions that trigger human Rpa2 phosphorylation, and its relation to other properties of damage responses. This also provides the opportunity to study Rpa2 NT modification in two different systems to gain an understanding of conserved properties and to potentially study the significance of differences in the damage response between the two systems.Support or Funding InformationThis research was supported by NSF‐CAREER 1253723 to SJH.

Molecular Evolution and Functional Diversification of Replication Protein A1 in Plants.

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding complex required for eukaryotic DNA replication, repair, and recombination. RPA is composed of three subunits, RPA1, RPA2, and RPA3. In contrast to single RPA subunit genes generally found in animals and yeast, plants encode multiple paralogs of RPA subunits, suggesting subfunctionalization. Genetic analysis demonstrates that five Arabidopsis thaliana RPA1 paralogs (RPA1A to RPA1E) have unique and overlapping functions in DNA replication, repair, and meiosis. We hypothesize here that RPA1 subfunctionalities will be reflected in major structural and sequence differences among the paralogs. To address this, we analyzed amino acid and nucleotide sequences of RPA1 paralogs from 25 complete genomes representing a wide spectrum of plants and unicellular green algae. We find here that the plant RPA1 gene family is divided into three general groups termed RPA1A, RPA1B, and RPA1C, which likely arose from two progenitor groups in unicellular green algae. In the family Brassicaceae the RPA1B and RPA1C groups have further expanded to include two unique sub-functional paralogs RPA1D and RPA1E, respectively. In addition, RPA1 groups have unique domains, motifs, cis-elements, gene expression profiles, and pattern of conservation that are consistent with proposed functions in monocot and dicot species, including a novel C-terminal zinc-finger domain found only in plant RPA1C-like sequences. These results allow for improved prediction of RPA1 subunit functions in newly sequenced plant genomes, and potentially provide a unique molecular tool to improve classification of Brassicaceae species.

Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination.

We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage.

Genetic Analysis of Replication Protein A Large Subunit Family in Arabidopsis Reveals its Role in the DNA Damage Response Pathway

Replication Protein A (RPA) is a heterotrimeric single stranded binding protein required for eukaryotic DNA replication, repair and recombination. The primary biochemical function of RPA is to protect and preserve single stranded DNA from nucleolytic degradation and hairpin formation. Downstream cellular responses to detect DNA damage include regulation of cell‐cycle checkpoints, induction of DNA repair and programmed cell death. These responses are mediated through two closely related protein kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia Telangiectasia‐mutated and Rad3‐Related). ATM is activated by double strand breaks while ATR is activated by a variety of lesions resulting in stalled replication forks. Genetic analysis of RPA1, by employing T‐DNA insertion mutants of the five Arabidopsis RPA1 subunit genes, revealed divergence of the RPA1 gene family into three functionally distinct groups. RPA1B and RPA1D are required for normal DNA replication, RPA1A and RPA1C play a role in meiosis, and RPA1C and RPA1E are primarily involved in DNA repair. Our analysis shows that within the repair group individual RPA1 subunits display unique functions in response to DNA damage. Analysis of hypersensitivity responses to replication blocking agents and DNA damaging agents suggests that RPA subunits RPA1C and RPA1E play leading roles in DNA damage repair. Genetic interactions of RPA1C, RPA1E and ATR revealed that a combination of rpa1c, rpa1e and atr mutants create additive hypersensitivity phenotypes consistent with each functioning in unique repair pathways.

Identification of stroke-associated-antigens via screening of recombinant proteins from the human expression cDNA library (SEREX).

BackgroundBecause circulating antibodies against a variety of antigens have been detected in patients with coronary heart disease, carotid atherosclerosis and those who have suffered a stroke, it is suspected that immune response may be one of the mechanisms of atherogenesis The objective of this study is to identify novel antibodies in ischemic stroke patients by screening the expressed recombinant proteins using a human cDNA library (SEREX).MethodsTo identify the candidate antigens, cDNA library was screened by SEREX using plasma from ten patients with ischemic stroke. Subsequently, via ELISA using recombinant proteins and synthetic peptides, the serum antibody levels were measured in two independent patient/healthy donor (HD) cohorts (142 and 78 in the 2nd screening and a validation cohort, respectively).ResultsThe initial screening resulted in the identification of six candidate antigens. Of these antigens, replication protein A2 (RPA2) was determined to be the antigen associated with stroke (P < 0.05) by ELISA with 2nd screening and validation cohort. Multifactorial logistic regression analysis showed that the increased levels of the RPA2 antibodies (RPA2-Abs) associated with stroke independent of other risk factors for stroke (P < 0.05). Receiver operating curve analysis demonstrated that the area under the curve from ELISA using GST fusion RPA2 and synthetic peptides (bRPA2-132) were 0.867 (95% CI: 0.798-0.936) and 0.971 (95% CI: 0.940-1.00), respectively. If the cut-off value of the bRPA2-132-Ab level was determined to be 0.334, the sensitivity and specificity of the antibody level as the diagnostic marker for stroke were 0.323 (95% CI: 0.209-0.453) and 1.00 (95% CI: 0.713-1.00), respectively.ConclusionsSEREX identified RPA2 as the antigen associated with ischemic stroke and serum auto-antibodies against RPA2 elevates in stroke patients. RPA2-Abs could become a biomarker for the evaluation of ischemic stroke at risk.

The DNA damage response and checkpoint adaptation in Saccharomyces cerevisiae: distinct roles for the replication protein A2 (Rfa2) N-terminus.

In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with “repairable” DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a “last-resort” mechanism for cell survival.

Raltitrexed's effect on the development of neural tube defects in mice is associated with DNA damage, apoptosis, and proliferation.

The causal metabolic pathway and the underlying mechanism between folate deficiency and neural tube defects (NTDs) remain obscure. Thymidylate (dTMP) is catalyzed by thymidylate synthase (TS) using the folate-derived one-carbon unit as the sole methyl donor. This study aims to examine the role of dTMP biosynthesis in the development of neural tube in mice by inhibition of TS via a specific inhibitor, raltitrexed (RTX). Pregnant mice were intraperitoneally injected with various doses of RTX on gestational day 7.5, and embryos were examined for the presence of NTDs on gestational day 11.5. TS activity and changes of dUMP and dTMP levels were measured following RTX treatment at the optimal dose. DNA damage was determined by detection of phosphorylated replication protein A2 (RPA2) and γ-H2AX in embryos with NTDs induced by RTX. Besides, apoptosis and proliferation were also analyzed in RTX-treated embryos with NTDs. We found that NTDs were highly occurred by the treatment of RTX at the optimal dose of 11.5 mg/kg b/w. RTX treatment significantly inhibited TS activity. Meanwhile, dTMP was decreased associated with the accumulation of dUMP in RTX-treated embryos. Phosphorylated RPA2 and γ-H2AX were significantly increased in RTX-treated embryos with NTDs compared to control. More apoptosis and decreased proliferation were also found in embryos with NTDs induced by RTX. These results indicate that impairment of dTMP biosynthesis caused by RTX led to the development of NTDs in mice. DNA damage and imbalance between apoptosis and proliferation may be potential mechanisms.

Replication Protein A2c Coupled with Replication Protein A1c Regulates Crossover Formation during Meiosis in Rice

Replication protein A (RPA) is a conserved heterotrimeric protein complex comprising RPA1, RPA2, and RPA3 subunits involved in multiple DNA metabolism pathways attributable to its single-stranded DNA binding property. Unlike other species possessing a single RPA2 gene, rice (Oryza sativa) possesses three RPA2 paralogs, but their functions remain unclear. In this study, we identified RPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (rpa2c) exhibited reduced bivalent formation, leading to chromosome nondisjunction. In rpa2c, chiasma frequency is reduced by ~78% compared with the wild type and is accompanied by loss of the obligate chiasma. The residual ~22% chiasmata fit a Poisson distribution, suggesting loss of crossover control. RPA2c colocalized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of RPA2c to the chromosomes. In addition, RPA2c partially colocalized with MER3 during late leptotene, thus indicating that RPA2c is required for class I crossover formation at a late stage of homologous recombination. Furthermore, we identified RPA1c, an RPA1 subunit with nearly overlapping distribution to RPA2c, required for ~79% of chiasmata formation. Our results demonstrate that an RPA complex comprising RPA2c and RPA1c is required to promote meiotic crossovers in rice.

Protein interaction and cellular localization of human CDC45

CDC45, which plays a role in eukaryotic DNA replication, is a member of the CMG (CDC45/MCM2-7/GINS) complex that is thought to function as a replicative DNA helicase. However, the biochemical properties of CDC45 are not fully understood. We systematically examined the interactions of human CDC45 with MCM2-7, GINS and other replication proteins by immunoprecipitation. We found that CDC45 can directly interact with all MCM2-7 proteins; with PSF2, PSF3 and SLD5 in GINS subunits; and with replication protein A2 (RPA2), AND-1 and topoisomerase 2-binding protein 1. These results are consistent with the notion that CDC45 plays a role in progression of DNA replication forks. Experiments using antibodies against CDC45 show that the level of CDC45 recovered from the Triton-insoluble chromatin-containing fraction is peaked at middle of S phase in synchronized HeLa cells. However, incubation of the Triton-insoluble fraction with nucleases resulted in recovery of less than half the amount of CDC45 in the nuclease-sensitive fraction; this result is in contrast with RPA1 and proliferating cell nuclear antigen distribution. These results indicate that a considerable portion of CDC45 localizes in a region other than the DNA replication forks in nuclei or it localizes on the replication forks but it is not fractionated with the fork proteins owing to its tight association with presumably nuclear scaffolds.

4E-BP3 regulates eIF4E-mediated nuclear mRNA export and interacts with replication protein A2

In nucleus, eIF4E regulates the nucleus export of specific mRNA. In this study, altered 4E-BP3 (eIF4E-binding protein 3) expression resulted in profoundly affected cyclin D1 protein levels, partially due to changes in the cytoplasmic cyclin D1 mRNA levels in both U2OS and MCF7 cells, whereas altered 4E-BP1 expression did not affect eIF4E-mediated cyclin D1 mRNA export. 4E-BP3 also affected a subset of growth promoting mRNAs exported in an eIF4-dependent manner. Furthermore, 4E-BP3 interacted with dephosphorylated RPA2 (replication protein A2). The results indicated 4E-BP3 acts as an inhibitor of eIF4E-mediated mRNA export in the examined cells, and 4E-BP3 inhibition of eIF4E-mediated mRNA export is regulated by the phosphorylation state of RPA2. Structured summary of protein interactions4EBP3 physically interacts with RPA2 by anti bait coimmunoprecipitation (View interaction)

Fludarabine Nucleoside Modulates Nuclear “Survival and Death” Proteins in Resistant Chronic Lymphocytic Leukemia Cells

The nuclear mechanisms by which fludarabine nucleoside (F-ara-A) induces apoptosis have been investigated in human MEC1 cells derived from B-cell chronic lymphocytic leukemia. Upon treatment of cells with F-ara-A (100 μM, 72 hours), 15 nuclear proteins changed in abundance by more than 2-fold. Nuclear proteins up-regulated included calmodulin (4.3-fold), prohibitin (3.9-fold), β-actin variant (3.7-fold), and structure-specific recognition protein 1 (3.7-fold); those down-regulated included 60S ribosomal protein P2B (0.12-fold), fumarate hydratase (0.19-fold), splicing factor arginine/serine-rich 3 (0.35-fold), and replication protein A2 (0.42-fold). These changes in the levels of specific proteins promote survival or apoptosis; because the end result is apoptosis of MEC1 cells, apoptotic effects predominate.

Activation of DNA damage response pathways in human mesenchymal stem cells exposed to cisplatin or γ-irradiation

DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to γ-irradiation and cisplatin, key DNA damage responses have been characterised in hMSCs from normal adult donors. Cisplatin and γ-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, γ-H2AX staining decreased by 24 hours following gamma-irradiation. γ-irradiation arrested hMSCs in the G1 phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and γ-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared to peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and γ-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment.

RPA2 (replication protein A2, 32kDa)

Review on RPA2 (replication protein A2, 32kDa), with data on DNA, on the protein encoded, and where the gene is implicated.

Increased RPA1 Gene Dosage Affects Genomic Stability Potentially Contributing to 17p13.3 Duplication Syndrome

A novel microduplication syndrome involving various-sized contiguous duplications in 17p13.3 has recently been described, suggesting that increased copy number of genes in 17p13.3, particularly PAFAH1B1, is associated with clinical features including facial dysmorphism, developmental delay, and autism spectrum disorder. We have previously shown that patient-derived cell lines from individuals with haploinsufficiency of RPA1, a gene within 17p13.3, exhibit an impaired ATR-dependent DNA damage response (DDR). Here, we show that cell lines from patients with duplications specifically incorporating RPA1 exhibit a different although characteristic spectrum of DDR defects including abnormal S phase distribution, attenuated DNA double strand break (DSB)-induced RAD51 chromatin retention, elevated genomic instability, and increased sensitivity to DNA damaging agents. Using controlled conditional over-expression of RPA1 in a human model cell system, we also see attenuated DSB-induced RAD51 chromatin retention. Furthermore, we find that transient over-expression of RPA1 can impact on homologous recombination (HR) pathways following DSB formation, favouring engagement in aberrant forms of recombination and repair. Our data identifies unanticipated defects in the DDR associated with duplications in 17p13.3 in humans involving modest RPA1 over-expression.

Replication protein A: a reliable biologic marker of prognostic and therapeutic value in human astrocytic tumors

Replication protein A is a single-stranded DNA-binding protein that is required for the stabilization of single-stranded DNA and identified in replication foci where members of cyclin-dependent kinases-cyclin complexes are also present. In this study, we investigated the expression of replication protein A1 and replication protein A2 subunits of replication protein A protein in correlation with cyclins D2 and D3 and nuclear factor κB expression and assessed their prognostic significance in 66 patients with astrocytomas. Statistically significant positive associations emerged between (a) replication protein A1 and replication protein A2 protein expression (P < .0001); (b) cyclins D2 and D3 expression (P < .0001); (c) replication protein A1, replication protein A2, and cyclins D2 and D3 expression and histologic grade (P = .0001 in all correlations); (d) replication protein A1 and cyclin D2 or D3 expression (P < .0001 in both relationships); and (e) replication protein A2 and cyclin D2 or D3 expression (P < .0001 in both relationships). Nuclear factor κB1/p50 expression was positively correlated with replication protein A1, replication protein A2, and cyclins D2 and D3 expression, although these relationships failed to retain statistical significance when they were adjusted for histologic grade. Replication protein A2 expression seemed to independently affect survival in grade IV (P = .005) as well as in the entire cohort (P = .006). None of the molecules under study seemed to influence survival in lower grades (II/III), either by univariate or by multivariate analysis. In conclusion, replication protein A1, replication protein A2, and cyclins D2 and D3 seem to have a parallel role in the promotion of cell cycle in astrocytic tumors being implicated in the malignant progression of these neoplasms. Moreover, replication protein A2 protein seems to be a useful prognostic indicator in patients with astrocytomas.

Functional Characterization of a Cancer Causing Mutation in Human Replication Protein A

Replication protein A (RPA) is the primary ssDNA-binding protein in eukaryotes. RPA is essential for DNA replication, repair, and recombination. Mutation of a conserved leucine residue to proline in the high-affinity DNA binding site of RPA (residue L221 in human RPA) has been shown to have defects in DNA repair and a high rate of chromosomal rearrangements in yeast. The homologous mutation in mice was found to be lethal when homozygous and to cause high rates of cancer when heterozygous. To understand the molecular defect causing these phenotypes, we created the homologous mutation in the human RPA1 gene (L221P) and analyzed its properties in cells and in vitro. RPA1(L221P) does not support cell cycle progression when it is the only form of RPA1 in HeLa cells. This phenotype is caused by defects in DNA replication and repair. No phenotype is observed when cells contain both wild-type and L221P forms of RPA1, indicating that L221P is not dominant. Recombinant L221P polypeptide forms a stable complex with the other subunits of RPA, indicating that the mutation does not destabilize the protein; however, the resulting complex has dramatically reduced ssDNA binding activity and cannot support SV40 DNA replication in vitro. These findings indicate that in mammals, the L221P mutation causes a defect in ssDNA binding and a nonfunctional protein complex. This suggests that haploinsufficiency of RPA causes an increase in the levels of DNA damage and in the incidence of cancer.

Transcription-dependent Activation of Ataxia Telangiectasia Mutated Prevents DNA-dependent Protein Kinase-mediated Cell Death in Response to Topoisomerase I Poison

Camptothecin (CPT) is a topoisomerase I inhibitor, derivatives of which are being used for cancer chemotherapy. CPT-induced DNA double-strand breaks (DSBs) are considered a major cause of its tumoricidal activity, and it has been shown that CPT induces DNA damage signaling through the phosphatidylinositol 3-kinase-related kinases, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase). In addition, CPT causes DNA strand breaks mediated by transcription, although the downstream signaling events are less well characterized. In this study, we show that CPT-induced activation of ATM requires transcription. Mechanistically, transcription inhibition suppressed CPT-dependent activation of ATM and blocked recruitment of the DNA damage mediator p53-binding protein 1 (53BP1) to DNA damage sites, whereas ATM inhibition abrogated CPT-induced G(1)/S and S phase checkpoints. Functional inactivation of ATM resulted in DNA replication-dependent hyperactivation of DNA-PK in CPT-treated cells and dramatic CPT hypersensitivity. On the other hand, simultaneous inhibition of ATM and DNA-PK partially restored CPT resistance, suggesting that activation of DNA-PK is proapoptotic in the absence of ATM. Correspondingly, comet assay and cell cycle synchronization experiments suggested that transcription collapse occurring as the result of CPT treatment are converted to frank double-strand breaks when ATM-deficient cells bypass the G(1)/S checkpoint. Thus, ATM suppresses DNA-PK-dependent cell death in response to topoisomerase poisons, a finding with potential clinical implications.

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

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.

Proteomic analysis of BRCA1-depleted cell line reveals a putative role for replication protein A2 up-regulation in BRCA1 breast tumor development

Germline mutations in BRCA1 result in a strong predisposition to breast cancer, with frequent loss of heterozygosity of the remaining wild-type allele. The development of BRCA1 tumors is likely to depend on additional genetic alterations and gene expression changes which follow growth and DNA repair defects associated with BRCA1 deficiency. The identification of these modifications offers an opportunity to find surrogate markers of BRCA1 tumors. Here, we sought to identify differentially expressed proteins related to BRCA1 depletion. We used isogenic HeLa cells either stably knocked-down or not for BRCA1 (BRCA1(KD) ) and compared protein profiles of these cells by DIGE. We detected increased levels of Replication protein A2 (RPA2) in BRCA1(KD) cells as compared to control cells. RPA2 is an essential protein required for DNA replication and repair. We further demonstrated that depletion of RPA2 subunit delays growth of BRCA1(KD) respect to isogenic control cells. Strikingly, elevated levels of RPA2 were more frequently observed in BRCA1 tumors when triple-negative tumors from BRCA1 mutation carriers (n=13) and non-carriers (n=36) were stained in situ for RPA2. RPA2 up-regulation may thus be involved in the growth and/or survival of BRCA1 tumor cells and useful in immunohistochemical discrimination of triple-negative BRCA1 tumors.

Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin

The ubiquitin–proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.