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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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Related in: MedlinePlus

Validation of TLS candidate genes by qPCR-based and colony-based TLS assays.(a) An outline of the qPCR-based TLS assay. In brief, following TLS in mammalian cells, the plasmids were extracted under alkaline conditions, and subsequently treated with single-stranded-specific endonuclease. This ensures that only filled-in plasmids remain intact. TLS efficiency was calculated as the ratio between the products of qPCRs targeting the filled-in gap-lesion plasmid, and the filled-in control gapped plasmid. (b) qPCR-based TLS assays in MEFs. Ten hit genes were examined for involvement in TLS across the tobacco smoke-induced lesion BP-G and the ultraviolet-induced damage TT 6-4 PP, with Rev3L, encoding the catalytic subunit of DNA polymerase-ζ, serving as a positive control. Data are represented as mean±s.e.m. of 4–6 biological replicas. One-sample t-test, P values are indicated as a colour code above each column. (c) mRNA knockdown efficiencies by the siRNAs used in b. Values were measured by qPCR and normalized to sample treated with control siRNA (siCont). Mean values±s.e.m. of three replicas are presented. (d) Colony-based TLS assay across BP-G in mouse cells. Data are represented as mean±s.e.m. of three biological replicas. All the tested genes gave two-tailed t-test, P values <0.02. (e) Mutagenicity of TLS as observed in the colony-based TLS assays shown in d. Ninety-six colonies were analysed per condition. χ2P values were calculated relative to control siRNA. ssDNA, single-stranded DNA.
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f3: Validation of TLS candidate genes by qPCR-based and colony-based TLS assays.(a) An outline of the qPCR-based TLS assay. In brief, following TLS in mammalian cells, the plasmids were extracted under alkaline conditions, and subsequently treated with single-stranded-specific endonuclease. This ensures that only filled-in plasmids remain intact. TLS efficiency was calculated as the ratio between the products of qPCRs targeting the filled-in gap-lesion plasmid, and the filled-in control gapped plasmid. (b) qPCR-based TLS assays in MEFs. Ten hit genes were examined for involvement in TLS across the tobacco smoke-induced lesion BP-G and the ultraviolet-induced damage TT 6-4 PP, with Rev3L, encoding the catalytic subunit of DNA polymerase-ζ, serving as a positive control. Data are represented as mean±s.e.m. of 4–6 biological replicas. One-sample t-test, P values are indicated as a colour code above each column. (c) mRNA knockdown efficiencies by the siRNAs used in b. Values were measured by qPCR and normalized to sample treated with control siRNA (siCont). Mean values±s.e.m. of three replicas are presented. (d) Colony-based TLS assay across BP-G in mouse cells. Data are represented as mean±s.e.m. of three biological replicas. All the tested genes gave two-tailed t-test, P values <0.02. (e) Mutagenicity of TLS as observed in the colony-based TLS assays shown in d. Ninety-six colonies were analysed per condition. χ2P values were calculated relative to control siRNA. ssDNA, single-stranded DNA.

Mentions: To further validate the hits, a second version of the TLS assay was developed based on quantitative PCR (qPCR) readouts rather than gene expression (Fig. 3a). In brief, following TLS in mammalian cells, the plasmids were extracted under alkaline conditions, which denatured gapped plasmids but not the covalently closed products of TLS or the control gap-filling reactions. Remnants of gapped plasmids were digested with S1 single-stranded endonuclease. TLS efficiency was calculated as the ratio between the products of qPCRs that targeted the filled-in gap-lesion plasmid and the filled-in control gapped plasmid, and was normalized to control siRNA-treated samples (Fig. 3a). The qPCR-based TLS assay was utilized to examine the involvement of 10 selected hits in TLS across the TT 6-4 PP ultraviolet lesion in mouse embryonic fibroblasts (MEFs). In parallel, TLS was measured across the major tobacco smoke-induced DNA adduct benzo[a]pyrene-guanine (BP-G), a non-ultraviolet lesion. Six hits, namely Papd7, Ruvbl2, Trip11, Npm1, Abh2 and Ube2e1, significantly affected TLS across both DNA lesions, although the effects obtained for Abh2 and Ube2e1 were rather small (Fig. 3b, t-test, P values <0.05; knockdown efficiencies are shown in Fig. 3c). Cyld, Mcm3, Dclre1a and Ercc4 affected TLS across TT 6-4 PP but not across BP-G (Fig. 3b, Cyld knockdown reduced TLS across BP-G, but gave a marginal P value of 0.06). Of notice, all the 10 tested human genes were validated in the qPCR-based TLS assay with at least one DNA lesion in MEFs. Four genes were further tested for their impact on TLS extent and mutagenicity in a gapped plasmid assay in which the readout is based on transformation of Escherichia coli cells, and therefore enables determining the sequence signature of TLS events173238. Knocking down the expression of each of the tested genes, namely Cyld, Npm1, Papd7 and Ruvbl2, significantly reduced TLS efficiency across the BP-G adduct (Fig. 3d; two-tailed t-test, P values <0.02). DNA sequence analysis showed that in control cells TLS was 47% accurate (insertion of a dCMP opposite BP-G), and 53% were point mutations, most of which (87%) were caused by insertion of dAMP opposite the lesion (Fig. 3e), consistent with previous results1739. Interestingly, knocking down the expression of each of the four tested genes also caused, in addition to the decrease in the extent of TLS, a lower error rate among the TLS events (Fig. 3e).


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)

Validation of TLS candidate genes by qPCR-based and colony-based TLS assays.(a) An outline of the qPCR-based TLS assay. In brief, following TLS in mammalian cells, the plasmids were extracted under alkaline conditions, and subsequently treated with single-stranded-specific endonuclease. This ensures that only filled-in plasmids remain intact. TLS efficiency was calculated as the ratio between the products of qPCRs targeting the filled-in gap-lesion plasmid, and the filled-in control gapped plasmid. (b) qPCR-based TLS assays in MEFs. Ten hit genes were examined for involvement in TLS across the tobacco smoke-induced lesion BP-G and the ultraviolet-induced damage TT 6-4 PP, with Rev3L, encoding the catalytic subunit of DNA polymerase-ζ, serving as a positive control. Data are represented as mean±s.e.m. of 4–6 biological replicas. One-sample t-test, P values are indicated as a colour code above each column. (c) mRNA knockdown efficiencies by the siRNAs used in b. Values were measured by qPCR and normalized to sample treated with control siRNA (siCont). Mean values±s.e.m. of three replicas are presented. (d) Colony-based TLS assay across BP-G in mouse cells. Data are represented as mean±s.e.m. of three biological replicas. All the tested genes gave two-tailed t-test, P values <0.02. (e) Mutagenicity of TLS as observed in the colony-based TLS assays shown in d. Ninety-six colonies were analysed per condition. χ2P values were calculated relative to control siRNA. ssDNA, single-stranded DNA.
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Related In: Results  -  Collection

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

f3: Validation of TLS candidate genes by qPCR-based and colony-based TLS assays.(a) An outline of the qPCR-based TLS assay. In brief, following TLS in mammalian cells, the plasmids were extracted under alkaline conditions, and subsequently treated with single-stranded-specific endonuclease. This ensures that only filled-in plasmids remain intact. TLS efficiency was calculated as the ratio between the products of qPCRs targeting the filled-in gap-lesion plasmid, and the filled-in control gapped plasmid. (b) qPCR-based TLS assays in MEFs. Ten hit genes were examined for involvement in TLS across the tobacco smoke-induced lesion BP-G and the ultraviolet-induced damage TT 6-4 PP, with Rev3L, encoding the catalytic subunit of DNA polymerase-ζ, serving as a positive control. Data are represented as mean±s.e.m. of 4–6 biological replicas. One-sample t-test, P values are indicated as a colour code above each column. (c) mRNA knockdown efficiencies by the siRNAs used in b. Values were measured by qPCR and normalized to sample treated with control siRNA (siCont). Mean values±s.e.m. of three replicas are presented. (d) Colony-based TLS assay across BP-G in mouse cells. Data are represented as mean±s.e.m. of three biological replicas. All the tested genes gave two-tailed t-test, P values <0.02. (e) Mutagenicity of TLS as observed in the colony-based TLS assays shown in d. Ninety-six colonies were analysed per condition. χ2P values were calculated relative to control siRNA. ssDNA, single-stranded DNA.
Mentions: To further validate the hits, a second version of the TLS assay was developed based on quantitative PCR (qPCR) readouts rather than gene expression (Fig. 3a). In brief, following TLS in mammalian cells, the plasmids were extracted under alkaline conditions, which denatured gapped plasmids but not the covalently closed products of TLS or the control gap-filling reactions. Remnants of gapped plasmids were digested with S1 single-stranded endonuclease. TLS efficiency was calculated as the ratio between the products of qPCRs that targeted the filled-in gap-lesion plasmid and the filled-in control gapped plasmid, and was normalized to control siRNA-treated samples (Fig. 3a). The qPCR-based TLS assay was utilized to examine the involvement of 10 selected hits in TLS across the TT 6-4 PP ultraviolet lesion in mouse embryonic fibroblasts (MEFs). In parallel, TLS was measured across the major tobacco smoke-induced DNA adduct benzo[a]pyrene-guanine (BP-G), a non-ultraviolet lesion. Six hits, namely Papd7, Ruvbl2, Trip11, Npm1, Abh2 and Ube2e1, significantly affected TLS across both DNA lesions, although the effects obtained for Abh2 and Ube2e1 were rather small (Fig. 3b, t-test, P values <0.05; knockdown efficiencies are shown in Fig. 3c). Cyld, Mcm3, Dclre1a and Ercc4 affected TLS across TT 6-4 PP but not across BP-G (Fig. 3b, Cyld knockdown reduced TLS across BP-G, but gave a marginal P value of 0.06). Of notice, all the 10 tested human genes were validated in the qPCR-based TLS assay with at least one DNA lesion in MEFs. Four genes were further tested for their impact on TLS extent and mutagenicity in a gapped plasmid assay in which the readout is based on transformation of Escherichia coli cells, and therefore enables determining the sequence signature of TLS events173238. Knocking down the expression of each of the tested genes, namely Cyld, Npm1, Papd7 and Ruvbl2, significantly reduced TLS efficiency across the BP-G adduct (Fig. 3d; two-tailed t-test, P values <0.02). DNA sequence analysis showed that in control cells TLS was 47% accurate (insertion of a dCMP opposite BP-G), and 53% were point mutations, most of which (87%) were caused by insertion of dAMP opposite the lesion (Fig. 3e), consistent with previous results1739. Interestingly, knocking down the expression of each of the four tested genes also caused, in addition to the decrease in the extent of TLS, a lower error rate among the TLS events (Fig. 3e).

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