Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States
Journal of Cell Science (Impact Factor: 5.43). 01/2007; 119(Pt 24):5137-46. DOI: 10.1242/jcs.03291
Source: PubMed


Werner syndrome (WS) is a rare genetic disorder characterized by genomic instability caused by defects in the WRN gene encoding a member of the human RecQ helicase family. RecQ helicases are involved in several DNA metabolic pathways including homologous recombination (HR) processes during repair of stalled replication forks. Following introduction of interstrand DNA crosslinks (ICL), WRN relocated from nucleoli to arrested replication forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, we use fluorescence resonance energy transfer (FRET) and immune-precipitation experiments to demonstrate that WRN participates in a multiprotein complex including RAD51, RAD54, RAD54B and ATR in cells where replication has been arrested by ICL. We verify the WRN-RAD51 and WRN-RAD54B direct interaction in vitro. Our data support a role for WRN also in the recombination step of ICL repair.

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    • "ID1, D2, D3 and IA1, A2, A3 were determined for cells transfected with CFP and YFP constructs only, with same settings and same fluorescence intensities as co-transfected cells (I1, I2, I3). FRET >0 was normalized for expression levels using the equation: NFRET = FRET/(I1×I3)1/2 [20], [21]. NFRET was calculated from mean intensities within a region of interest representing replication foci containing more than 25 pixels where all pixels had intensities below 250. "
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    ABSTRACT: Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.
    PLoS ONE 07/2013; 8(7):e70430. DOI:10.1371/journal.pone.0070430 · 3.23 Impact Factor
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    • "Also the Werner protein (WRN) has been linked to DNA repair when ICLs cause replication fork breakdown (Otterlei et al., 2006). WRN has a recombination Q (RecQ) helicase and an exonuclease domain and functions during replication and recombination repair. "
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    ABSTRACT: DNA damage contributes to cancer development and aging. Congenital syndromes that affect DNA repair processes are characterized by cancer susceptibility, developmental defects, and accelerated aging (Schumacher et al., 2008). DNA damage interferes with DNA metabolism by blocking replication and transcription. DNA polymerase blockage leads to replication arrest and can gives rise to genome instability. Transcription, on the other hand, is an essential process for utilizing the information encoded in the genome. DNA damage that interferes with transcription can lead to apoptosis and cellular senescence. Both processes are powerful tumor suppressors (Bartek and Lukas, 2007). Cellular response mechanisms to stalled RNA polymerase II complexes have only recently started to be uncovered. Transcription-coupled DNA damage responses might thus play important roles for the adjustments to DNA damage accumulation in the aging organism (Garinis et al., 2009). Here we review human disorders that are caused by defects in genome stability to explore the role of DNA damage in aging and disease. We discuss how the nucleotide excision repair system functions at the interface of transcription and repair and conclude with concepts how therapeutic targeting of transcription might be utilized in the treatment of cancer.
    Frontiers in Genetics 02/2013; 4:19. DOI:10.3389/fgene.2013.00019
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    • "FRET was scored when the intensity of emitted light from YFP after excitation of the CFP fluorochrome was stronger than the light emitted by CFP or YFP-tagged proteins alone, after excitation with CFP lasers (false FRET), given by the equation: FRET =  I2– I1 (ID2/ID1) - I3 (IA2/IA3). FRET >0 was normalized for expression levels using the equation: NFRET  =  FRET/(I1 × I3)1/2 [45], [46]. NFRET was calculated from mean intensities (I) within a region of interest (ROI) containing more than 25 pixels where all pixels had intensities below 250. "
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    ABSTRACT: Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA(-/-) cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6-4) photoproducts, and are consequently more arrested in S phase as compared to XPA(-/-) cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.
    PLoS ONE 11/2012; 7(11):e49199. DOI:10.1371/journal.pone.0049199 · 3.23 Impact Factor
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