RPA1

Gene Summary

Gene:RPA1; replication protein A1
Aliases: HSSB, RF-A, RP-A, REPA1, RPA70, MST075
Location:17p13.3
Summary:-
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:replication protein A 70 kDa DNA-binding subunit
Source:NCBIAccessed: 11 March, 2017

Ontology:

What does this gene/protein do?
Show (39)
Pathways:What pathways are this gene/protein implicaed in?
Show (1)

Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 11 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Protein Binding
  • Cancer Gene Expression Regulation
  • DNA-Binding Proteins
  • Cultured Cells
  • Skin Cancer
  • Lung Cancer
  • Oligonucleotide Array Sequence Analysis
  • DNA Repair
  • RPA1
  • HeLa Cells
  • Transcription Factors
  • Cell Cycle Proteins
  • Chromosome 17
  • Cell Proliferation
  • Xeroderma Pigmentosum Group A Protein
  • Molecular Sequence Data
  • Zinc Fingers
  • DNA Replication
  • Cancer DNA
  • Nuclear Proteins
  • Thymine
  • DNA Damage
  • Cell Cycle
  • p53 Protein
  • Western Blotting
  • siRNA
  • Up-Regulation
  • Genetic Predisposition
  • Replication Protein A
  • Gene Expression Profiling
  • Mutation
  • Antineoplastic Agents
  • Cell Line
  • Base Sequence
  • Ultraviolet Rays
  • Breast Cancer
  • DNA Helicases
  • DNA, Single-Stranded
  • Recombinant Proteins
  • DNA
  • Kinetics
Tag cloud generated 11 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (3)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: RPA1 (cancer-related)

Chang ET, Parekh PR, Yang Q, et al.
Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells.
Oncotarget. 2016; 7(9):10578-93 [PubMed] Free Access to Full Article Related Publications
The heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by coordinating the translation of selected transcripts associated with proliferation and survival. hnRNP A18 binds to and stabilizes the transcripts of pro-survival genes harboring its RNA signature motif in their 3'UTRs. hnRNP A18 binds to ATR, RPA, TRX, HIF-1α and several protein translation factor mRNAs on polysomes and increases de novo protein translation under cellular stress. Most importantly, down regulation of hnRNP A18 decreases proliferation, invasion and migration in addition to significantly reducing tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, tissue microarrays performed on human melanoma, prostate, breast and colon cancer indicate that hnRNP A18 is over expressed in 40 to 60% of these malignant tissue as compared to normal adjacent tissue. Immunohistochemistry data indicate that hnRNP A18 is over expressed in the stroma and hypoxic areas of human tumors. These data thus indicate that hnRNP A18 can promote tumor growth in in vivo models by coordinating the translation of pro-survival transcripts to support the demands of proliferating cells and increase survival under cellular stress. hnRNP A18 therefore represents a new target to selectively inhibit protein translation in tumor cells.

Panero J, Stella F, Schutz N, et al.
Differential Expression of Non-Shelterin Genes Associated with High Telomerase Levels and Telomere Shortening in Plasma Cell Disorders.
PLoS One. 2015; 10(9):e0137972 [PubMed] Free Access to Full Article Related Publications
Telomerase, shelterin proteins and various interacting factors, named non-shelterin proteins, are involved in the regulation of telomere length (TL). Altered expression of any of these telomere-associated genes can lead to telomere dysfunction, causing genomic instability and disease development. In this study, we investigated the expression profile of a set of non-shelterin genes involved in essential processes such as replication (RPA1), DNA damage repair pathways (MRE11-RAD50-NBS1) and stabilization of telomerase complex (DKC1), in 35 patients with monoclonal gammopathy of undetermined significance (MGUS) and 40 cases with multiple myeloma (MM). Results were correlated with hTERT expression, TL and clinical parameters. Overall, a significant increase in DKC1, RAD50, MRE11, NBS1 and RPA1 expression along with an upregulation of hTERT in MM compared with MGUS was observed (p≤0.032). Interestingly, in both entities high mRNA levels of non-shelterin genes were associated with short TLs and increased hTERT expression. Significant differences were observed for DKC1 in MM (p ≤0.026), suggesting an important role for this gene in the maintenance of short telomeres by telomerase in myeloma plasma cells. With regard to clinical associations, we observed a significant increase in DKC1, RAD50, MRE11 and RPA1 expression in MM cases with high bone marrow infiltration (p≤0.03) and a tendency towards cases with advanced ISS stage, providing the first evidence of non-shelterin genes associated to risk factors in MM. Taken together, our findings bring new insights into the intricate mechanisms by which telomere-associated proteins collaborate in the maintenance of plasma cells immortalization and suggest a role for the upregulation of these genes in the progression of the disease.

Flynn RL, Cox KE, Jeitany M, et al.
Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors.
Science. 2015; 347(6219):273-7 [PubMed] Free Access to Full Article Related Publications
Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin-remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric noncoding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. The cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.

Smith SC, Petrova AV, Madden MZ, et al.
A gemcitabine sensitivity screen identifies a role for NEK9 in the replication stress response.
Nucleic Acids Res. 2014; 42(18):11517-27 [PubMed] Free Access to Full Article Related Publications
The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.

Patterson MJ, Sutton RE, Forrest I, et al.
Assessing the function of homologous recombination DNA repair in malignant pleural effusion (MPE) samples.
Br J Cancer. 2014; 111(1):94-100 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells.
METHODS: A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of γH2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells.
RESULTS: From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples - three NSCLC and one mesothelioma - were HRR defective and eight samples - one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers - were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1.
CONCLUSIONS: HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi.

Di Z, Sanyuan S, Hong L, Dahai Y
Enhanced radiosensitivity and G2/M arrest were observed in radioresistant esophageal cancer cells by knocking down RPA expression.
Cell Biochem Biophys. 2014; 70(2):887-91 [PubMed] Related Publications
The aim of this study was to evaluate the changes in radiosensitivity of radioresistant esophageal cancer cells (TE-1R) after disruption of replication protein A (RPA) expression and to explore the potential mechanism. A radioresistant human esophageal cancer cell line TE-1R was established by treating TE-1 cells with the radiation. Then, siRPA1 or -2 was transfected to TE-1R cells. The untransfected group (control) and nonsense short interfering RNA (siRNA) transfected group (NC) were used as controls. To investigate the radiosensitivity changes of TE-1R cells, the dose-survival curve was established by colony-forming assay, and the cell cycle distribution was measured by flow cytometry. (1) Comparing with control and NC groups, the protein expression of RPA1 and -2 decreased significantly 48 h after siRPA1 or -2 transfection. (2) The D 0, D q, and SF2 values reduced from 2.09, 1.70, and 0.85 in NC group to 1.67, 0.71, and 0.44 and 1.82, 0.89, and 0.51 in siRNA1 and siRPA2 transfected groups, respectively. The D q sensitization enhancement ratios (SERDq) were 2.39 and 1.91 in siRNA1 and siRPA2 transfected groups, respectively. (3) The G2/M arrest was significantly caused by siRPA1 or -2 transfection as compared with that in the NC group (t value was 2.827, 2.853, p < 0.05). Post transcriptional silencing of RPA1 or -2 via RNAi can enhance the radiosensitivity of human esophageal cancer cells TE-1R, and the potential mechanism may be related to the inhibition of post-radiation sublethal damage repair and the halted cell cycle progression at G2/M phase. Therefore, RPA may become a new target for radiosensitization enhancement in esophageal cancer.

Hoxha M, Fabris S, Agnelli L, et al.
Relevance of telomere/telomerase system impairment in early stage chronic lymphocytic leukemia.
Genes Chromosomes Cancer. 2014; 53(7):612-21 [PubMed] Related Publications
Several studies have proposed telomere length and telomerase activity as prognostic factors in chronic lymphocytic leukemia (CLL), whereas information addressing the role of telomere-associated genes is limited. We measured relative telomere length (RTL) and TERT expression levels in purified peripheral CD19(+) B-cells from seven healthy donors and 77 untreated CLLs in early stage disease (Binet A). Data were correlated with the major biological and cytogenetic markers, global DNA methylation (Alu and LINE-1), and clinical outcome. The expression profiles of telomere-associated genes were also investigated. RTL was decreased in CLLs as compared with controls (P < 0.001); within CLL, a progressive and significant RTL shortening was observed in patients from 13q- through +12, 11q-, and 17p- alterations; short telomeres were significantly associated with unmutated IGHV configuration and global DNA hypomethylation. Decreased RTL was associated with a shorter time to first treatment. A significant upregulation of POT1, TRF1, RAP1, MRE11A, RAD50, and RPA1 transcript levels was observed in CLLs compared with controls. Our study suggests that impairment of telomere/telomerase system represents an early event in CLL pathogenesis. Moreover, the correlation between telomere shortening and global DNA hypomethylation supports the involvement of DNA hypomethylation to increase chromosome instability. © 2014 Wiley Periodicals, Inc.

Ma Z, Bi Q, Wang Y
Hydrogen sulfide accelerates cell cycle progression in oral squamous cell carcinoma cell lines.
Oral Dis. 2015; 21(2):156-62 [PubMed] Related Publications
OBJECTIVE: To investigate the cell cycle regulator role of the third gaseous transmitter hydrogen sulfide (H2 S) in three oral SCC cell lines by using NaHS, a donor of H2 S.
METHODS: The synchronized oral squamous cell carcinoma cell lines (Cal27, GNM, and WSU-HN6) were treated with different concentrations of NaHS and then subjected to cell proliferation, cell cycle, and Western blot analyses.
RESULTS: The CCK-8 assay results showed that the exogenously administered H2 S donor, NaHS, induced CAL-27, and GNM cell proliferation in a concentration-dependent manner, and the cell cycle analysis indicated that NaHS accelerated cell cycle progression of the synchronized CAL-27, GNM, and WSU-HN6 cells. Western blot analysis revealed that the cell cycle regulatory genes RPA70 and RB1 were significantly down-regulated and that proliferating cell nuclear antigen (PCNA) and CDK4 were markedly up-regulated by NaHS at specific time points in the cell cycle. In addition, our results indicated that the phosphorylation of Akt and Erk1/2 was involved in exogenous H2 S-induced oral SCC cell proliferation.
CONCLUSIONS: H2 S is a potential pro-proliferative factor of human oral SCC cells that accelerates the progression of the SCC cell cycle; thus, H2 S plays a deleterious role in oral SCC cancer development.

Won KJ, Im JY, Yun CO, et al.
Human Noxin is an anti-apoptotic protein in response to DNA damage of A549 non-small cell lung carcinoma.
Int J Cancer. 2014; 134(11):2595-604 [PubMed] Related Publications
Human Noxin (hNoxin, C11Orf82), a homolog of mouse noxin, is highly expressed in colorectal and lung cancer tissues. hNoxin contains a DNA-binding C-domain in RPA1, which mediates DNA metabolic processes, such as DNA replication and DNA repair. Expression of hNoxin is associated with S phase in cancer cells and in normal cells. Expression of hNoxin was induced by ultraviolet (UV) irradiation. Knockdown of hNoxin caused growth inhibition of colorectal and lung cancer cells. The comet assay and western blot analysis revealed that hNoxin knockdown induced apoptosis through activation of p38 mitogen-activated protein kinase (MAPK)/p53 in non-small cell lung carcinoma A549 cells. Furthermore, simultaneous hNoxin knockdown and treatment with DNA-damaging agents, such as camptothecin (CPT) and UV irradiation, enhanced apoptosis, whereas Trichostatin A (TSA) did not. However, transient overexpression of hNoxin rescued cells from DNA damage-induced apoptosis but did not block apoptosis in the absence of DNA damage. These results suggest that hNoxin may be associated with inhibition of apoptosis in response to DNA damage. An adenovirus expressing a short hairpin RNA against hNoxin transcripts significantly suppressed the growth of A549 tumor xenografts, indicating that hNoxin knockdown has in vivo anti-tumor efficacy. Thus, hNoxin is a DNA damage-induced anti-apoptotic protein and potential therapeutic target in cancer.

Liu M, Wu R, Yang F, et al.
Identification of FN1BP1 as a novel cell cycle regulator through modulating G1 checkpoint in human hepatocarcinoma Hep3B cells.
PLoS One. 2013; 8(2):e57574 [PubMed] Free Access to Full Article Related Publications
A novel human gene, FN1BP1 (fibronectin 1 binding protein 1), was identified using the human placenta cDNA library. Northern blotting showed a transcript of ∼2.8 kb in human placenta, liver, and skeletal muscle tissues. This mRNA transcript length was similar to the full FN1BP1 sequence obtained previously. We established a conditionally induced stable cell line of Hep3B-Tet-on-FN1BP1 to investigate the preliminary function and mechanism of the secretory FN1BP1 protein. Cell-proliferation and colony-conformation assays demonstrated that FN1BP1 protein suppressed Hep3B cell growth and colonization in vitro. Analysis of Atlas human cDNA expression indicated that after FN1BP1 Dox-inducing expression for 24 h, 19 genes were up-regulated and 22 genes were down-regulated more than two-fold. Most of these gene changes were related to cell-cycle-arrest proteins (p21cip1, p15, and cyclin E1), transcription factors (general transcription factors, zinc finger proteins, transcriptional enhancer factors), SWI/SNF (SWItch/Sucrose NonFermentable) complex units, early-response proteins, and nerve growth or neurotrophic factors. Down-regulated genes were subject to colony-stimulating factors (e.g., GMSFs), and many repair genes were involved in DNA damage (RAD, ERCC, DNA topoisomerase, polymerase, and ligase). Some interesting genes (p21cip1, ID2, GMSF, ERCC5, and RPA1), which changed in the cDNA microarray analysis, were confirmed by semi-qRT-PCR, and similar changes in expression were observed. FCM cell-cycle analysis indicated that FN1BP1 over-expression could result in G1 phase arrest. FN1BP1 might inhibit cell growth and/or colony conformation through G1 phase arrest of the Hep3B cell cycle. These results indicate the potential role of FN1BP1 as a treatment target for hepatocellular carcinoma.

Maute RL, Schneider C, Sumazin P, et al.
tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma.
Proc Natl Acad Sci U S A. 2013; 110(4):1404-9 [PubMed] Free Access to Full Article Related Publications
Sequencing studies from several model systems have suggested that diverse and abundant small RNAs may be derived from tRNA, but the function of these molecules remains undefined. Here, we demonstrate that one such tRNA-derived fragment, cloned from human mature B cells and designated CU1276, in fact possesses the functional characteristics of a microRNA, including a DICER1-dependent biogenesis, physical association with Argonaute proteins, and the ability to repress mRNA transcripts in a sequence-specific manner. Expression of CU1276 is abundant in normal germinal center B cells but absent in germinal center-derived lymphomas, suggesting a role in the pathogenesis of this disease. Furthermore, CU1276 represses endogenous RPA1, an essential gene involved in many aspects of DNA dynamics, and consequently, expression of this tRNA-derived microRNA in a lymphoma cell line suppresses proliferation and modulates the molecular response to DNA damage. These results establish that functionally active microRNAs can be derived from tRNA, thus defining a class of genetic entities with potentially important biological roles.

Calle ML, Urrea V, Boulesteix AL, Malats N
AUC-RF: a new strategy for genomic profiling with random forest.
Hum Hered. 2011; 72(2):121-32 [PubMed] Related Publications
OBJECTIVE: Genomic profiling, the use of genetic variants at multiple loci simultaneously for the prediction of disease risk, requires the selection of a set of genetic variants that best predicts disease status. The goal of this work was to provide a new selection algorithm for genomic profiling.
METHODS: We propose a new algorithm for genomic profiling based on optimizing the area under the receiver operating characteristic curve (AUC) of the random forest (RF). The proposed strategy implements a backward elimination process based on the initial ranking of variables.
RESULTS AND CONCLUSIONS: We demonstrate the advantage of using the AUC instead of the classification error as a measure of predictive accuracy of RF. In particular, we show that the use of the classification error is especially inappropriate when dealing with unbalanced data sets. The new procedure for variable selection and prediction, namely AUC-RF, is illustrated with data from a bladder cancer study and also with simulated data. The algorithm is publicly available as an R package, named AUCRF, at http://cran.r-project.org/.

Martrat G, Maxwell CM, Tominaga E, et al.
Exploring the link between MORF4L1 and risk of breast cancer.
Breast Cancer Res. 2011; 13(2):R40 [PubMed] Free Access to Full Article Related Publications
INTRODUCTION: Proteins encoded by Fanconi anemia (FA) and/or breast cancer (BrCa) susceptibility genes cooperate in a common DNA damage repair signaling pathway. To gain deeper insight into this pathway and its influence on cancer risk, we searched for novel components through protein physical interaction screens.
METHODS: Protein physical interactions were screened using the yeast two-hybrid system. Co-affinity purifications and endogenous co-immunoprecipitation assays were performed to corroborate interactions. Biochemical and functional assays in human, mouse and Caenorhabditis elegans models were carried out to characterize pathway components. Thirteen FANCD2-monoubiquitinylation-positive FA cell lines excluded for genetic defects in the downstream pathway components and 300 familial BrCa patients negative for BRCA1/2 mutations were analyzed for genetic mutations. Common genetic variants were genotyped in 9,573 BRCA1/2 mutation carriers for associations with BrCa risk.
RESULTS: A previously identified co-purifying protein with PALB2 was identified, MRG15 (MORF4L1 gene). Results in human, mouse and C. elegans models delineate molecular and functional relationships with BRCA2, PALB2, RAD51 and RPA1 that suggest a role for MRG15 in the repair of DNA double-strand breaks. Mrg15-deficient murine embryonic fibroblasts showed moderate sensitivity to γ-irradiation relative to controls and reduced formation of Rad51 nuclear foci. Examination of mutants of MRG15 and BRCA2 C. elegans orthologs revealed phenocopy by accumulation of RPA-1 (human RPA1) nuclear foci and aberrant chromosomal compactions in meiotic cells. However, no alterations or mutations were identified for MRG15/MORF4L1 in unclassified FA patients and BrCa familial cases. Finally, no significant associations between common MORF4L1 variants and BrCa risk for BRCA1 or BRCA2 mutation carriers were identified: rs7164529, Ptrend = 0.45 and 0.05, P2df = 0.51 and 0.14, respectively; and rs10519219, Ptrend = 0.92 and 0.72, P2df = 0.76 and 0.07, respectively.
CONCLUSIONS: While the present study expands on the role of MRG15 in the control of genomic stability, weak associations cannot be ruled out for potential low-penetrance variants at MORF4L1 and BrCa risk among BRCA2 mutation carriers.

Wu Q, Beland FA, Chang CW, Fang JL
XPC is essential for nucleotide excision repair of zidovudine-induced DNA damage in human hepatoma cells.
Toxicol Appl Pharmacol. 2011; 251(2):155-62 [PubMed] Related Publications
Zidovudine (3'-azido-3'-dexoythymidine, AZT), a nucleoside reverse transcriptase inhibitor, can be incorporated into DNA and cause DNA damage. The mechanisms underlying the repair of AZT-induced DNA damage are unknown. To investigate the pathways involved in the recognition and repair of AZT-induced DNA damage, human hepatoma HepG2 cells were incubated with AZT for 2 weeks and the expression of DNA damage signaling pathways was determined using a pathway-based real-time PCR array. Compared to control cultures, damaged DNA binding and nucleotide excision repair (NER) pathways showed significantly increased gene expression. Further analysis indicated that AZT treatment increased the expression of genes associated with NER, including XPC, XPA, RPA1, GTF2H1, and ERCC1. Western blot analysis demonstrated that the protein levels of XPC and GTF2H1 were also significantly up-regulated. To explore further the function of XPC in the repair of AZT-induced DNA damage, XPC expression was stably knocked down by 71% using short hairpin RNA interference. In the XPC knocked-down cells, 100 μM AZT treatment significantly increased [³H]AZT incorporation into DNA, decreased the total number of viable cells, increased the release of lactate dehydrogenase, induced apoptosis, and caused a more extensive G2/M cell cycle arrest when compared to non-transfected HepG2 cells or HepG2 cells transfected with a scrambled short hairpin RNA sequence. Overall, these data indicate that XPC plays an essential role in the NER repair of AZT-induced DNA damage.

Vaid M, Sharma SD, Katiyar SK
Proanthocyanidins inhibit photocarcinogenesis through enhancement of DNA repair and xeroderma pigmentosum group A-dependent mechanism.
Cancer Prev Res (Phila). 2010; 3(12):1621-9 [PubMed] Related Publications
Dietary grape seed proanthocyanidins (GSP) inhibit photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated. As ultraviolet B (UVB)-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) has been implicated in skin cancer risk, we studied whether dietary GSPs enhance repair of UVB-induced DNA damage and, if so, what is the potential mechanism? Supplementation of GSPs (0.5%, w/w) with AIN76A control diet significantly reduced the levels of CPD(+) cells in UVB-exposed mouse skin; however, GSPs did not significantly reduce UVB-induced CPD(+) cells in the skin of interleukin-12p40 (IL-12) knockout (KO) mice, suggesting that IL-12 is required for the repair of CPDs by GSPs. Using IL-12 KO mice and their wild-type counterparts and standard photocarcinogenesis protocol, we found that supplementation of control diet with GSPs (0.5%, w/w) significantly reduced UVB-induced skin tumor development in wild-type mice, which was associated with the elevated mRNA levels of nucleotide excision repair genes, such as XPA, XPC, DDB2, and RPA1; however, this effect of GSPs was less pronounced in IL-12 KO mice. Cytostaining analysis revealed that GSPs repaired UV-induced CPD(+) cells in xeroderma pigmentosum complementation group A (XPA)-proficient fibroblasts from a healthy individual but did not repair in XPA-deficient fibroblasts from XPA patients. Furthermore, GSPs enhance nuclear translocation of XPA and enhanced its interactions with other DNA repair protein ERCC1. Together, our findings reveal that prevention of photocarcinogenesis by GSPs is mediated through enhanced DNA repair in epidermal cells by IL-12- and XPA-dependent mechanisms.

Guillem VM, Cervantes F, Martínez J, et al.
XPC genetic polymorphisms correlate with the response to imatinib treatment in patients with chronic phase chronic myeloid leukemia.
Am J Hematol. 2010; 85(7):482-6 [PubMed] Related Publications
Chronic myeloid leukemia (CML) is driven by the BCR-ABL protein, which promotes the proliferation and viability of the leukemic cells. Moreover, BCR-ABL induces genomic instability that can contribute to the emergence of resistant clones to the ABL kinase inhibitors. It is currently unknown whether the inherited individual capability to repair DNA damage could affect the treatment results. To address this, a comprehensive analysis of single nucleotide polymorphisms (SNPs) on the nucleotide excision repair (NER) genes (ERCC2-ERCC8, RPA1-RPA3, LIG1, RAD23B, XPA, XPC) was performed in 92 chronic phase CML patients treated with imatinib upfront. ERCC5 and XPC SNPs correlated with the response to imatinib. Haplotype analysis of XPC showed that the wild-type haplotype (499C-939A) was associated with a better response to imatinib. Moreover, the 5-year failure free survival for CA carriers was significantly better than that of the non-CA carriers (98% vs. 73%; P = 0.02). In the multivariate logistic model with genetic data and clinical covariates, the hemoglobin (Hb) level and the XPC haplotype were independently associated with the treatment response, with patients having a Hb < or =11 g/dl (Odds ratio [OR] = 5.0, 95% confidence interval [CI] = 1.5-16.1) or a non-CA XPC haplotype (OR = 4.1, 95% CI = 1.6-10.6) being at higher risk of suboptimal response/treatment failure. Our findings suggest that genetic polymorphisms in the NER pathway may influence the results to imatinib treatment in CML.

Guggenheim ER, Xu D, Zhang CX, et al.
Photoaffinity isolation and identification of proteins in cancer cell extracts that bind to platinum-modified DNA.
Chembiochem. 2009; 10(1):141-57 [PubMed] Free Access to Full Article Related Publications
The activity of the anticancer drug cisplatin is a consequence of its ability to bind DNA. Platinum adducts bend and unwind the DNA duplex, creating recognition sites for nuclear proteins. Following DNA damage recognition, the lesions will either be repaired, facilitating cell viability, or if repair is unsuccessful and the Pt adduct interrupts vital cellular functions, apoptosis will follow. With the use of the benzophenone-modified cisplatin analogue Pt-BP6, 25 bp DNA duplexes containing either a 1,2-d(G*pG*) intrastrand or a 1,3-d(G*pTpG*) intrastrand crosslink were synthesized, where the asterisks designate platinated nucleobases. Proteins having affinity for these platinated DNAs were photocrosslinked and identified in cervical, testicular, pancreatic and bone cancer-cell nuclear extracts. Proteins identified in this manner include the DNA repair factors RPA1, Ku70, Ku80, Msh2, DNA ligase III, PARP-1, and DNA-PKcs, as well as HMG-domain proteins HMGB1, HMGB2, HMGB3, and UBF1. The latter strongly associate with the 1,2-d(G*pG*) adduct and weakly or not at all with the 1,3-d(G*pTpG*) adduct. The nucleotide excision repair protein RPA1 was photocrosslinked only by the probe containing a 1,3-d(G*pTpG*) intrastrand crosslink. The affinity of PARP-1 for platinum-modified DNA was established using this type of probe for the first time. To ensure that the proteins were not photocrosslinked because of an affinity for DNA ends, a 90-base dumbbell probe modified with Pt-BP6 was investigated. Photocrosslinking experiments with this longer probe revealed the same proteins, as well as some additional proteins involved in chromatin remodeling, transcription, or repair. These findings reveal a more complete list of proteins involved in the early steps of the mechanism of action of the cisplatin and its close analogue carboplatin than previously was available.

Nishiwaki T, Kobayashi N, Iwamoto T, et al.
Comparative study of nucleotide excision repair defects between XPD-mutated fibroblasts derived from trichothiodystrophy and xeroderma pigmentosum patients.
DNA Repair (Amst). 2008; 7(12):1990-8 [PubMed] Related Publications
To get a clue to understand how mutations in the XPD gene result in different skin cancer susceptibilities in patients with xeroderma pigmentosum (XP) or trichothiodystrophy (TTD), a thorough understanding of their nucleotide excision repair (NER) defects is essential. Here, we extensively characterize the possible causes of NER defects in XP-D and in TTD fibroblasts. The 3 XP-D cell strains examined were similarly deficient in repairing UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) from genomic DNA. The severity of NER defects correlated with their UV sensitivities. Possible alterations of TFIIH (which consists of 10 subunits including XPD) were then examined. All XP-D cell strains were normal in their concentrations of TFIIH, and displayed normal abilities to recruit TFIIH to sites of UV-induced DNA damage. However, replication protein A (RPA; single-stranded DNA binding protein) accumulation at DNA damage sites, which probably reflects the in vivo XPD helicase activity of TFIIH, is similarly impaired in all XP-D cell strains. Meanwhile, all 3 TTD cell strains had approximately 50% decreases in cellular TFIIH content. Importantly, 2 of the 3 TTD cell strains, which carry the major XPD mutations found in TTD patients, showed defective recruitment of TFIIH to DNA damage sites. Moreover, RPA accumulation at damage sites was impaired in all TTD cell strains to different degrees, which correlated with the severity of their NER defects. These results demonstrate that XP-D and TTD cells are both deficient in the repair of CPDs and 6-4PPs, but TTD cells have more multiple causes for their NER defects than do XP-D cells. Since TFIIH is a repair/transcription factor, TTD-specific alterations of TFIIH possibly result in transcriptional defects, which might be implication for the lack of increased incidence of skin cancers in TTD patients.

Ogawa R, Ishiguro H, Kuwabara Y, et al.
Identification of candidate genes involved in the radiosensitivity of esophageal cancer cells by microarray analysis.
Dis Esophagus. 2008; 21(4):288-97 [PubMed] Related Publications
Radiotherapy plays a key role in the control of tumor growth in esophageal cancer patients. To identify the patients who will benefit most from radiation therapy, it is important to know the genes that are involved in the radiosensitivity of esophageal cancer cells. Hence, we examined the global gene expression in radiosensitive and radioresistant esophageal squamous cell carcinoma cell lines. Radiosensitivities of 13 esophageal cancer cell lines were measured. RNA was extracted from each esophageal cancer cell line and a normal esophageal epithelial cell line, and the global gene expression profiles were analyzed using a 34 594-spot oligonucleotide microarray. In the clonogenic assay, one cell line (TE-11) was identified to be highly sensitive to radiation, while the other cell lines were found to be relatively radioresistant. We identified 71 candidate genes that were differentially expressed in TE-11 by microarray analysis. The up-regulated genes included CABPR, FABP5, DSC2, GPX2, NME, CBR3, DOCK8, and ABCC5, while the down-regulated genes included RPA1, LDOC1, NDN, and SKP1A. Our investigation provided comprehensive information on genes related to radiosensitivity of esophageal cancer cells; this information can serve as a basis for further functional studies.

Vermeer MH, van Doorn R, Dijkman R, et al.
Novel and highly recurrent chromosomal alterations in Sézary syndrome.
Cancer Res. 2008; 68(8):2689-98 [PubMed] Related Publications
This study was designed to identify highly recurrent genetic alterations typical of Sézary syndrome (Sz), an aggressive cutaneous T-cell lymphoma/leukemia, possibly revealing pathogenetic mechanisms and novel therapeutic targets. High-resolution array-based comparative genomic hybridization was done on malignant T cells from 20 patients. Expression levels of selected biologically relevant genes residing within loci with frequent copy number alteration were measured using quantitative PCR. Combined binary ratio labeling-fluorescence in situ hybridization karyotyping was done on malignant cells from five patients. Minimal common regions with copy number alteration occurring in at least 35% of patients harbored 15 bona fide oncogenes and 3 tumor suppressor genes. Based on the function of the identified oncogenes and tumor suppressor genes, at least three molecular mechanisms are relevant in the pathogenesis of Sz. First, gain of cMYC and loss of cMYC antagonists (MXI1 and MNT) were observed in 75% and 40% to 55% of patients, respectively, which were frequently associated with deregulated gene expression. The presence of cMYC/MAX protein heterodimers in Sézary cells was confirmed using a proximity ligation assay. Second, a region containing TP53 and genome maintenance genes (RPA1/HIC1) was lost in the majority of patients. Third, the interleukin 2 (IL-2) pathway was affected by gain of STAT3/STAT5 and IL-2 (receptor) genes in 75% and 30%, respectively, and loss of TCF8 and DUSP5 in at least 45% of patients. In sum, the Sz genome is characterized by gross chromosomal instability with highly recurrent gains and losses. Prominent among deregulated genes are those encoding cMYC, cMYC-regulating proteins, mediators of MYC-induced apoptosis, and IL-2 signaling pathway components.

Poncet D, Belleville A, t'kint de Roodenbeke C, et al.
Changes in the expression of telomere maintenance genes suggest global telomere dysfunction in B-chronic lymphocytic leukemia.
Blood. 2008; 111(4):2388-91 [PubMed] Related Publications
In this study, we explored the telomeric changes that occur in B-chronic lymphocytic leukemia (B-CLL), in which telomere length has recently been demonstrated to be a powerful prognostic marker. We carried out a transcriptomic analysis of telomerase components (hTERT and DYSKERIN), shelterin proteins (TRF1, TRF2, hRAP1, TIN2, POT1, and TPP1), and a set of multifunctional proteins involved in telomere maintenance (hEST1A, MRE11, RAD50, Ku80, and RPA1) in peripheral B cells from 42 B-CLL patients and 20 healthy donors. We found that, in B-CLL cells, the expressions of hTERT, DYSKERIN, TRF1, hRAP1, POT1, hEST1A, MRE11, RAD50, and KU80 were more than 2-fold reduced (P < .001), contrasting with the higher expression of TPP1 and RPA1 (P < .001). This differential expression pattern suggests that both telomerase down-regulation and changes in telomeric proteins composition are involved in the pathogenesis of B-CLL.

Michiels S, Danoy P, Dessen P, et al.
Polymorphism discovery in 62 DNA repair genes and haplotype associations with risks for lung and head and neck cancers.
Carcinogenesis. 2007; 28(8):1731-9 [PubMed] Related Publications
DNA repair is essential for the maintenance of genetic stability. We undertook sequencing to determine common genetic variants in 70 genes involved in three major repair pathways (base excision repair, nucleotide excision repair and mismatch repair) and in DNA synthesis, and investigated their relationship to lung and head and neck (H-N) cancers. Of the 70 genes examined, 62 were successfully screened (exon coverage >20%) by sequencing exons, parts of introns and flanking regions in 32 DNA samples from healthy Caucasian individuals. The strategy used allowed the detection of almost all variants with a minor allele frequency >or=5% in the regions sequenced. During single-nucleotide polymorphism (SNP) discovery, 772 sequences were detected in introns or regions flanking the gene and 313 were found in exons (leading to 113 non-synonymous variations) during single-nucleotide polymorphism (SNP) discovery. In total, 695 variants were successfully genotyped in 151 lung cancer cases, 251 H-N cancer cases and 172 hospital controls. Score statistics were used to test differences in haplotype frequencies between cases and controls in an unconditional logistic regression model. To account for multiple testing, we associated to each P-value an estimated proportion of false discoveries. Haplotype analysis revealed potential associations (P < 0.05) between lung cancer and eight genes (MSH3, MLH3, POLK, LIG1, ERCC5, PMS1, POLG2 and RPA3) and between H-N cancer and four genes (PMS1, POLG2, POLR2B and RPA1) with false discovery proportions of 25 and 55%, respectively. The DNA synthesis pathway showed a tendency for more differential SNP allele frequencies between H-N cases and controls than expected by chance (P = 0.05). These results hint to a few potential candidates for further investigation in larger studies.

Lapouge G, Millon R, Muller D, et al.
Cisplatin-induced genes as potential markers for thyroid cancer.
Cell Mol Life Sci. 2005; 62(1):53-64 [PubMed] Related Publications
Despite the uncontested role of p53 in cycle arrest/cell death after cisplatin treatment, to date the question whether wild-type p53 confers a resistant or sensitive status on the cell is still a matter of debate. Isogenic and isophenotypic human thyroid papillary carcinoma cell line variants for p53 differently expressed cycle genes after cisplatin treatment. Seven genes (CDC6-related protein, CCNC, GAS1, TFDP2, MAPK10/JNK3, WEE1, RPA1) selected after expression on an Atlas human cell cycle array were analyzed by quantitative real-time PCR. While cisplatin treatment increased their expression in p53 wild-type cells it decreased it in cells with inactivated p53 and had no or less effect on cells with mutated p53. These results show that in a well-defined system, different alterations of p53 can lead to a different regulation of genes and hence to either resistance or sensitivity to cisplatin. Moreover for the first time, MAPK10/JNK3 was identified in human thyroid cells and tissue. Four of the genes (CDC6-related protein, CCNC, GAS1 and TFDP2) were decreased in human papillary carcinoma tissues. Relevance of these genes (especially a decrease in GAS1 in thyroid papillary carcinoma) in various malignant pathologies has already been shown. These genes may be explored as new markers in advanced thyroid cancer such as metastatic and anaplastic forms displaying p53 alterations.

Gwynn B, Martina JA, Bonifacino JS, et al.
Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex.
Blood. 2004; 104(10):3181-9 [PubMed] Related Publications
Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.

Rochlitz C, Figlin R, Squiban P, et al.
Phase I immunotherapy with a modified vaccinia virus (MVA) expressing human MUC1 as antigen-specific immunotherapy in patients with MUC1-positive advanced cancer.
J Gene Med. 2003; 5(8):690-9 [PubMed] Related Publications
BACKGROUND: The MUC1 protein is a highly glycosylated mucin normally found at the apical surface of mucin-secreting epithelial cells in many types of tissues. MUC1 is expressed, but heavily underglycosylated, in different human tumors. TG4010 is a viral suspension of a recombinant vaccinia vector (MVA) containing DNA sequences coding for the human MUC1 antigen and interleukin-2 (IL-2). This product was developed for use as a vaccine in cancer patients whose tumors express the MUC1 antigen. The objective of the present study was to determine the safety of the product and to define the dose of TG4010 to be used in further clinical trials.
MATERIALS AND METHODS: Thirteen patients with different solid tumors were treated by repeated intramuscular injection with increasing doses of TG4010 in two separate phase I studies, one in Europe (Basel-CR) and one in the United States (UCLA-RF): a total of 6 patients were treated at a dose of 5 x 10(6) pfu, 3 patients at 5 x 10(7) pfu, and 4 patients at 10(8) pfu. Safety, efficacy, and different immunological tests were the endpoints of the study.
RESULTS: Tolerance of TG4010 was excellent, and side effects mainly consisted of injection site pain and influenza-like symptoms. There was no apparent detrimental effect of repeated injections of the vaccinia virus. Four of thirteen evaluable patients showed stabilization of their disease for 6 to 9 months. One lung cancer patient who was initially progressing after the first injections later showed a marked decrease in the size of his metastases that lasted for 14 months. Some T cell proliferative immune responses were seen in five patients.
CONCLUSIONS: The administration of TG4010 was generally well tolerated in patients with metastatic tumors, and transient disease stabilization was observed in several patients, warranting further clinical studies with the product.

Wong JM, Ionescu D, Ingles CJ
Interaction between BRCA2 and replication protein A is compromised by a cancer-predisposing mutation in BRCA2.
Oncogene. 2003; 22(1):28-33 [PubMed] Related Publications
Mutations in the BRCA1 and BRCA2 genes predispose women to familial, early-onset breast cancer. Both the BRCA1 and BRCA2 proteins appear to function in the homologous recombination pathway of DNA double-strand break repair. Both BRCA1 and BRCA2 have also been implicated in transcription by RNA polymerase II, for both proteins have domains which, when tethered adjacent to a promoter, can activate transcription. In experiments reported here, we have used protein affinity chromatography and coimmunoprecipitation techniques to show that the putative N-terminal acidic transcriptional activation domain of BRCA2 interacts with replication protein A (RPA), a protein essential for DNA repair, replication and recombination. This interaction was not mediated by DNA and was specific for human RPA but not yeast RPA. Since the cancer-predisposing mutation Y42C in BRCA2 significantly compromised the interaction between RPA and BRCA2, this interaction may be biologically important. That BRCA2 protein in HeLa cell extract also coimmunoprecipitated with RPA suggested that this interaction occurs in vivo. Therefore, the transcriptional activation domains within BRCA2, and perhaps BRCA1, may provide links to RPA and DNA repair processes rather than transcription.

Sukhodolets KE, Hickman AB, Agarwal SK, et al.
The 32-kilodalton subunit of replication protein A interacts with menin, the product of the MEN1 tumor suppressor gene.
Mol Cell Biol. 2003; 23(2):493-509 [PubMed] Free Access to Full Article Related Publications
Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.

Tomkiel JE, Alansari H, Tang N, et al.
Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer.
Clin Cancer Res. 2002; 8(3):752-8 [PubMed] Related Publications
PURPOSE: We sought to identify autoantigens recognized by antibodies in breast cancer patient sera with potential diagnostic or prognostic significance.
EXPERIMENTAL DESIGN: Serum from a female breast cancer patient exhibiting a high titer antinuclear antibody was used to screen a HeLa cDNA expression library, leading to the cloning of a cDNA for the M(r) 32,000 subunit of replication protein A (RPA32). RPA32 expression and localization were assayed in autologous tumor by monoclonal antibody staining. A specific ELISA using recombinant protein was used to screen sera from 801 breast cancer patients and 65 controls.
RESULTS: A relationship between anti-replication protein A (RPA) antibodies and the ductal breast carcinoma of the proband was suggested by overexpression and aberrant localization of RPA32 in tumor cells as compared with surrounding normal ductal tissue and by the presence of anti-RPA32 antibodies before the diagnosis. The prevalence of anti-RPA32 antibodies was significantly higher (P < 0.01) among breast cancer patients (87 of 801 patients) than among noncancer controls (0 of 65 controls). Similarly, anti-RPA32 antibodies were present in 4 of 39 patients with intraductal in situ carcinoma. No associations were found between anti-RPA antibodies and survival, occurrence of a second tumor, metastases, or antibodies to p53. Reactivity to RPA32 also was detected in sera from 3 of 47 patients with other cancers.
CONCLUSIONS: In view of the central role of RPA in DNA replication, recombination, and repair, we suggest that autoimmunity to RPA32 may reflect molecular changes involved in the process of tumorigenesis. The finding of antibodies to RPA32 before diagnosis and their prevalence in in situ carcinoma suggest that they are potentially useful markers of early disease.

Riva F, Zuco V, Vink AA, et al.
UV-induced DNA incision and proliferating cell nuclear antigen recruitment to repair sites occur independently of p53-replication protein A interaction in p53 wild type and mutant ovarian carcinoma cells.
Carcinogenesis. 2001; 22(12):1971-8 [PubMed] Related Publications
The tumour suppressor gene TP53 plays an important role in the regulation of DNA repair, and particularly of nucleotide excision repair. The influence of p53 status on the efficiency of the principal steps of this repair pathway was investigated after UV-C irradiation in the human ovarian carcinoma cell line IGROV-1 (expressing wild-type p53) and in the derived clone IGROV-1/Pt1 (with p53 mutations at codons 270 and 282). Clonogenic survival after UV-C irradiation showed that IGROV-1/Pt1 cells were approximately 2-fold more resistant to DNA damage than parental cells. Modulation of p53 protein levels, cell cycle arrest and apoptosis were induced in UV-irradiated IGROV-1 cells, but not in the p53-mutant cell line. Exposure to UV or cisplatin induced down-regulation of p53-replication protein A (RPA) interaction in parental, but not in IGROV-1/Pt1 cells. However, persistent binding of p53 to RPA did not affect the early steps of DNA repair. In fact, both UV-induced DNA incision and the recruitment of proliferating cell nuclear antigen (PCNA) to DNA repair sites occurred to a comparable extent in p53-wild type and -mutant cell lines, although PCNA remained associated with chromatin for a longer period of time in IGROV-1/Pt1 cells. Global genome repair, as detected by immunoblot analysis of cyclobutane pyrimidine dimers, was not significantly different in the two cell lines at 3 h after UV irradiation. In contrast, lesion removal at 24 h was markedly reduced in IGROV-1/Pt1 cells, being approximately 25% of the initial amount of damage, as compared with approximately 50% repair in parental cells. These results indicate that the presence of mutant p53 protein and its persistent interaction with RPA do not affect the early steps of nucleotide excision repair in IGROV-1/Pt1 cells. Thus, repair defects in p53-mutant ovarian carcinoma cells may be attributed to late events, possibly related to a reduced removal/recycling of PCNA at repair sites.

Oakley GG, Loberg LI, Yao J, et al.
UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein.
Mol Biol Cell. 2001; 12(5):1199-213 [PubMed] Free Access to Full Article Related Publications
Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

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