Douglas L. Pittman

The splicing-factor related protein SFPQ/PSF interacts with RAD51D and is necessary for homology-directed repair and sister chromatid cohesion

DNA double-stranded breaks (DSBs) are among the most severe forms of DNA damage and responsible for chromosomal translocations that may lead to gene fusions. The RAD51 family plays an integral role in preserving genome stability by homology directed repair of DSBs. From a proteomics screen, we recently identified SFPQ/PSF as an interacting partner with the RAD51 paralogs, RAD51D, RAD51C and XRCC2. Initially discovered as a potential RNA splicing factor, SFPQ was later shown to have homologous recombination and non-homologous end joining related activities and also to bind and modulate the function of RAD51. Here, we demonstrate that SFPQ interacts directly with RAD51D and that deficiency of both proteins confers a severe loss of cell viability, indicating a synthetic lethal relationship. Surprisingly, deficiency of SFPQ alone also leads to sister chromatid cohesion defects and chromosome instability. In addition, SFPQ was demonstrated to mediate homology directed DNA repair and DNA damage response resulting from DNA crosslinking agents, alkylating agents and camptothecin. Taken together, these data indicate that SFPQ association with the RAD51 protein complex is essential for homologous recombination repair of DNA damage and maintaining genome integrity.

Uracil in DNA: consequences for carcinogenesis and chemotherapy.

The synthesis of thymidylate (TMP) occupies a convergence of two critical metabolic pathways: folate metabolism and pyrimidine biosynthesis. Thymidylate is formed from deoxyuridylate (dUMP) using N(5),N(10)-methylene tetrahydrofolate. The metabolic relationship between dUMP, TMP, and folate has been the subject of cancer research from prevention to chemotherapy. Thymidylate stress is induced by nutritional deficiency of folic acid, defects in folate metabolism, and by antifolate and fluoropyrimidine chemotherapeutics. Both classes of chemotherapeutics remain mainstay treatments against solid tumors. Because of the close relationship between dUMP and TMP, thymidylate stress is associated with increased incorporation of uracil into DNA. Genomic uracil is removed by uracil DNA glycosylases of base excision repair (BER). Unfortunately, BER is apparently problematic during thymidylate stress. Because BER requires a DNA resynthesis step, elevated dUTP causes reintroduction of genomic uracil. BER strand break intermediates are clastogenic if not repaired. Thus, BER during thymidylate stress appears to cause genome instability, yet might also contribute to the mechanism of action for antifolates and fluoropyrimidines. However, the precise roles of BER and its components during thymidylate stress remain unclear. In particular, links between BER and downstream events remain poorly defined, including damage signaling pathways and homologous recombination (HR). Evidence is growing that HR responds to persistent BER strand break intermediates and DNA damage signaling pathways mediate cross talk between BER and HR. Examination of crosstalk among BER, HR, and damage signaling may shed light on decades of investigation and provide insight for development of novel chemopreventive and chemotherapeutic approaches.

The interaction profile of homologous recombination repair proteins RAD51C, RAD51D and XRCC2 as determined by proteomic analysis.

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.

Functional characterization and identification of mouse Rad51d splice variants

BackgroundThe homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns.ResultsYeast-2-hybrid analysis was used to determine that the Mus musculus Rad51d splice variant product RAD51DΔ7b (deleted for residues 219 through 223) was capable of interacting with both RAD51C and XRCC2 and that RAD51D+int3 interacted with XRCC2. In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2. Cellular localization, detected by EGFP fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. However, none of the splice variants were capable of restoring resistance of Rad51d-deficient cell lines to mitomycin C. RT-PCR expression analysis revealed that Rad51dΔ3 (deleted for exon 3) and Rad51dΔ5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dΔ3 being detected in each tissue except ovary and Rad51dΔ5 not detected in mammary gland and testis. These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10.ConclusionThese results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.

Cisplatin resistance conferred by the RAD51D (E233G) genetic variant is dependent upon p53 status in human breast carcinoma cell lines

RAD51D, a paralog of the mammalian RAD51 gene, contributes towards maintaining genomic integrity by homologous recombination DNA repair and telomere maintenance. A RAD51D variant, E233G, was initially identified as a potential susceptibility allele in high‐risk, site‐specific, familial breast cancer. We describe in this report that the Rad51d (E233G) genetic variant confers increased cisplatin resistance and cell growth phenotypes in human breast carcinoma cell lines with a mutant p53 gene (BT20 and T47D) but not with a wild‐type p53 gene (MCF‐7). Treatment with a p53 specific inhibitor, pifithrin α, restored this resistant phenotype in the MCF‐7 cell line. Additionally, Rad51d (E233G) conferred increased cisplatin resistance of an MCF7 cell line in which p53 expression was stably knocked down by shRNAp53, indicating that the effect of this variant is dependent upon p53 status. Further study of Rad51d (E233G) will provide mechanistic insight towards the role of RAD51D in cellular response to anticancer agents and as a potential target for cancer therapy.

Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

Abstract Purpose: Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock. Materials and methods: B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (γH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock. Results: Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the G2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged γH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR. Conclusions: The HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs.

The Homologous Recombination Protein RAD51D Mediates the Processing of 6-Thioguanine Lesions Downstream of Mismatch Repair

Thiopurines are extensively used as immunosuppressants and in the treatment of childhood cancers, even though there is concern about therapy-induced leukemias and myelodysplastic syndromes resulting from thiopurine use. Following metabolic activation, thiopurines are incorporated into DNA and invoke mismatch repair (MMR). Recognition of 6-thioguanine (6-thioG) in DNA by key MMR proteins results in cell death rather than repair. There are suggestions that homologous recombination (HR) is involved downstream of MMR following thiopurine treatment, but the precise role of HR is poorly understood. In this study, we demonstrate that cells deficient in RAD51D (a RAD51 paralogue) are extremely sensitive to 6-thioG. This sensitivity is almost completely rescued by the deletion of Mlh1, which suggests that HR is involved in the repair of the 6-thioG–induced recombinogenic lesions generated by MMR. Furthermore, 6-thioG induces chromosome aberrations in the Rad51d-deficient cells. Interestingly, Rad51d-deficient cells show a striking increase in the frequency of triradial and quadriradial chromosomes in response to 6-thioG therapy. The presence of these chromatid exchange–type aberrations indicates that the deficiency in RAD51D-dependent HR results in profound chromosomal damage precipitated by the processing of 6-thioG by MMR. The radials are notable as an important source of chromosomal translocations, which are the most common class of mutations found in hematologic malignancies. This study thus suggests that HR insufficiency could be a potential risk factor for the development of secondary cancers that result from long-term use of thiopurines in patients. Mol Cancer Res; 9(2); 206–14. ©2011 AACR.

RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity.

The RAD51 family is integral for homologous recombination (HR) mediated DNA repair and maintaining chromosome integrity. RAD51D, the fourth member of the family, is a known ovarian cancer susceptibility gene and required for the repair of interstrand crosslink DNA damage and preserving chromosomal stability. In this report, we describe the RNF138 E3 ubiquitin ligase that interacts with and ubiquitinates the RAD51D HR protein. RNF138 is a member of an E3 ligase family that contains an amino-terminal RING finger domain and a putative carboxyl-terminal ubiquitin interaction motif. In mammalian cells, depletion of RNF138 increased the stability of the RAD51D protein, suggesting that RNF138 governs ubiquitin-proteasome-mediated degradation of RAD51D. However, RNF138 depletion conferred sensitivity to DNA damaging agents, reduced RAD51 focus formation, and increased chromosomal instability. Site-specific mutagenesis of the RNF138 RING finger domain demonstrated that it was necessary for RAD51D ubiquitination. Presence of RNF138 also enhanced the interaction between RAD51D and a known interacting RAD51 family member XRCC2 in a yeast three-hybrid assay. Therefore, RNF138 is a newly identified regulatory component of the HR mediated DNA repair pathway that has implications toward understanding how ubiquitination modifies the functions of the RAD51 paralog protein complex.

Thiopurine‐induced mitotic catastrophe in Rad51d‐deficient mammalian cells

Thiopurines are part of a clinical regimen used for the treatment of autoimmune disorders and childhood acute lymphoblastic leukemia. However, despite these successes, there are also unintended consequences such as therapy‐induced cancer in long‐term survivors. Therefore, a better understanding of cellular responses to thiopurines will lead to improved and personalized treatment strategies. RAD51D is an important component of homologous recombination (HR), and our previous work established that mammalian cells defective for RAD51D are more sensitive to the thiopurine 6‐thioguanine (6TG) and have dramatically increased numbers of multinucleated cells and chromosome instability. 6TG is capable of being incorporated into telomeres, and interestingly, RAD51D contributes to telomere maintenance, although the precise function of RAD51D at the telomeres remains unclear. We sought here to investigate: (1) the activity of RAD51D at telomeres, (2) the contribution of RAD51D to protect against 6TG‐induced telomere damage, and (3) the fates of Rad51d‐deficient cells following 6TG treatment. These results demonstrate that RAD51D is required for maintaining the telomeric 3′ overhangs. As measured by γ‐H2AX induction and foci formation, 6TG induced DNA damage in Rad51d‐proficient and Rad51d‐deficient cells. However, the extent of γ‐H2AX telomere localization following 6TG treatment was higher in Rad51d‐deficient cells than in Rad51d‐proficient cells. Using live‐cell imaging of 6TG‐treated Rad51d‐deficient cells, two predominant forms of mitotic catastrophe were found to contribute to the formation of multinucleated cells, failed division and restitution. Collectively, these findings provide a unique window into the role of the RAD51D HR protein during thiopurine induction of mitotic catastrophe. Environ. Mol. Mutagen. 59:38–48, 2018.

Potential unintended consequences of getting rigorous with scientific rigor

Writing a counterpoint to the accompanying commentary seemed at first absurd. Arguing against rigor is likely itself considered to be unscientific. Striving for the absolute best technological, methodological and ethical practices when performing experiments will, of course, increase the likelihood of accuracy and reproducibility. This becomes even more important when performing clinical translational research that directly affects human lives. However, this latest fundamentalist movement, that is reflected in the accompanying opinion, to formalize and vigorously apply scientific rigor has potential long-term unintended consequences to constrain creativity and discovery. These issues go beyond the simple template changes in how US federal grant applications are formatted and the additional requirements for manuscript submissions. The concern about an ‘over-rigorization’ grows if it causes limitations of creative thinking needed to discover new foundational understandings and challenge established dogma. Every experimental design requires assumptions to test a hypothesis that may be proven incorrect. Just as important, it is necessary for science to retain the ability to retrospectively analyze and offer new interpretations of previously published data not flawed in its methodology, but possibly imperfect in its conclusions based on data limitations and models at that time. These issues are briefly discussed below in regards to time, complexity, selection pressures and fears.