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Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin.

Ziv O, Zeisel A, Mirlas-Neisberg N, Swain U, Nevo R, Ben-Chetrit N, Martelli MP, Rossi R, Schiesser S, Canman CE, Carell T, Geacintov NE, Falini B, Domany E, Livneh Z - Nat Commun (2014)

Bottom Line: We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη.Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη.These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a two-stage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

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Primary screen for ultraviolet sensitivity in NER-deficient human cells.(a) An outline of the primary screen. In brief, NER-deficient XPA cells were transfected with the siRNA libraries on day 0, exposed to ultraviolet C (UVC) irradiation (1 J m−2) on day 2 and analysed for cell viability on day 4. (b) Data reproducibility. Luminescence values of three biological replicas that were preformed on different days were plotted against each other. (c) Histogram describing ultraviolet-sensitivity fold change (FC) caused by the siRNAs (median values over the three replicas). Dashed red lines denote the distribution borders of negative control samples. (d) Enrichment of TLS, DNA repair and DNA-damage response pathways within the screen hits. Dashed red line corresponds to a hypergeometric test P value 0.05. NER, nucleotide excision repair; BER, base excision repair; MMR, mismatch repair; FA, the Fanconi anemia pathway; NHEJ, non-homologous end joining; HRR, homologous recombination repair; ATM and ATR, the two main DNA-damage response pathways. See also Supplementary Fig. 1, Supplementary Data 1 and Supplementary Table 1.
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f1: Primary screen for ultraviolet sensitivity in NER-deficient human cells.(a) An outline of the primary screen. In brief, NER-deficient XPA cells were transfected with the siRNA libraries on day 0, exposed to ultraviolet C (UVC) irradiation (1 J m−2) on day 2 and analysed for cell viability on day 4. (b) Data reproducibility. Luminescence values of three biological replicas that were preformed on different days were plotted against each other. (c) Histogram describing ultraviolet-sensitivity fold change (FC) caused by the siRNAs (median values over the three replicas). Dashed red lines denote the distribution borders of negative control samples. (d) Enrichment of TLS, DNA repair and DNA-damage response pathways within the screen hits. Dashed red line corresponds to a hypergeometric test P value 0.05. NER, nucleotide excision repair; BER, base excision repair; MMR, mismatch repair; FA, the Fanconi anemia pathway; NHEJ, non-homologous end joining; HRR, homologous recombination repair; ATM and ATR, the two main DNA-damage response pathways. See also Supplementary Fig. 1, Supplementary Data 1 and Supplementary Table 1.

Mentions: We performed a two-stage functional short interfering RNA (siRNA) screen designed to identify new mammalian TLS genes. In the first stage, we assayed ultraviolet sensitivity using an XPA cell line that is deficient in nucleotide excision repair (NER), and therefore defective in the repair of ultraviolet-induced DNA damage. Consequently, ultraviolet survival of the XPA cells exhibits a greater dependence on DNA-damage tolerance compared with NER-proficient cells38, making the screen more selective to DNA-damage tolerance genes. siRNAs that were identified in this stage as significantly affecting ultraviolet survival were re-screened with a second more stringent assay, which measured TLS. This strategy was used to screen 1,000 siRNAs directed to genes involved in DNA repair, ubiquitination and de-ubiquitination, cell cycle regulation and cancer. The ultraviolet-sensitivity screen (Fig. 1a) was performed in three biological replicas, exhibiting good reproducibility (Fig. 1b). Of the 1,000 genes assayed, we found 192 genes for which knockdown resulted in elevated ultraviolet sensitivity, and 45 genes that reduced ultraviolet sensitivity (false discovery rate (FDR) <8%; Fig. 1c, Supplementary Data 1, Supplementary Table 1 and Supplementary Fig. 1). Known TLS genes, as well as genes related to the ATR DNA-damage response pathway, but not other DNA repair pathways, were highly represented among the hits (Fig. 1d), suggesting that the XPA deficiency indeed enriched for genes involved in TLS.


Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin.

Ziv O, Zeisel A, Mirlas-Neisberg N, Swain U, Nevo R, Ben-Chetrit N, Martelli MP, Rossi R, Schiesser S, Canman CE, Carell T, Geacintov NE, Falini B, Domany E, Livneh Z - Nat Commun (2014)

Primary screen for ultraviolet sensitivity in NER-deficient human cells.(a) An outline of the primary screen. In brief, NER-deficient XPA cells were transfected with the siRNA libraries on day 0, exposed to ultraviolet C (UVC) irradiation (1 J m−2) on day 2 and analysed for cell viability on day 4. (b) Data reproducibility. Luminescence values of three biological replicas that were preformed on different days were plotted against each other. (c) Histogram describing ultraviolet-sensitivity fold change (FC) caused by the siRNAs (median values over the three replicas). Dashed red lines denote the distribution borders of negative control samples. (d) Enrichment of TLS, DNA repair and DNA-damage response pathways within the screen hits. Dashed red line corresponds to a hypergeometric test P value 0.05. NER, nucleotide excision repair; BER, base excision repair; MMR, mismatch repair; FA, the Fanconi anemia pathway; NHEJ, non-homologous end joining; HRR, homologous recombination repair; ATM and ATR, the two main DNA-damage response pathways. See also Supplementary Fig. 1, Supplementary Data 1 and Supplementary Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263322&req=5

f1: Primary screen for ultraviolet sensitivity in NER-deficient human cells.(a) An outline of the primary screen. In brief, NER-deficient XPA cells were transfected with the siRNA libraries on day 0, exposed to ultraviolet C (UVC) irradiation (1 J m−2) on day 2 and analysed for cell viability on day 4. (b) Data reproducibility. Luminescence values of three biological replicas that were preformed on different days were plotted against each other. (c) Histogram describing ultraviolet-sensitivity fold change (FC) caused by the siRNAs (median values over the three replicas). Dashed red lines denote the distribution borders of negative control samples. (d) Enrichment of TLS, DNA repair and DNA-damage response pathways within the screen hits. Dashed red line corresponds to a hypergeometric test P value 0.05. NER, nucleotide excision repair; BER, base excision repair; MMR, mismatch repair; FA, the Fanconi anemia pathway; NHEJ, non-homologous end joining; HRR, homologous recombination repair; ATM and ATR, the two main DNA-damage response pathways. See also Supplementary Fig. 1, Supplementary Data 1 and Supplementary Table 1.
Mentions: We performed a two-stage functional short interfering RNA (siRNA) screen designed to identify new mammalian TLS genes. In the first stage, we assayed ultraviolet sensitivity using an XPA cell line that is deficient in nucleotide excision repair (NER), and therefore defective in the repair of ultraviolet-induced DNA damage. Consequently, ultraviolet survival of the XPA cells exhibits a greater dependence on DNA-damage tolerance compared with NER-proficient cells38, making the screen more selective to DNA-damage tolerance genes. siRNAs that were identified in this stage as significantly affecting ultraviolet survival were re-screened with a second more stringent assay, which measured TLS. This strategy was used to screen 1,000 siRNAs directed to genes involved in DNA repair, ubiquitination and de-ubiquitination, cell cycle regulation and cancer. The ultraviolet-sensitivity screen (Fig. 1a) was performed in three biological replicas, exhibiting good reproducibility (Fig. 1b). Of the 1,000 genes assayed, we found 192 genes for which knockdown resulted in elevated ultraviolet sensitivity, and 45 genes that reduced ultraviolet sensitivity (false discovery rate (FDR) <8%; Fig. 1c, Supplementary Data 1, Supplementary Table 1 and Supplementary Fig. 1). Known TLS genes, as well as genes related to the ATR DNA-damage response pathway, but not other DNA repair pathways, were highly represented among the hits (Fig. 1d), suggesting that the XPA deficiency indeed enriched for genes involved in TLS.

Bottom Line: We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη.Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη.These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a two-stage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-η (polη), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of polη. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in ~30% of AML patients results in excessive degradation of polη. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1.

Show MeSH
Related in: MedlinePlus