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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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NPM1 protects DNA polη from proteasomal degradation.(a) MRC5sv cells stably expressing GFP-POLH were treated with siRNA against NPM1 or non- targeting control siRNA (siCont). GFP-polη levels were quantified by flow cytometry. (b,c) Immunoblot of polη extracted from human XPA cells (b) and MRC5sv cells (c) pretreated with siRNA against NPM1 or non-targeting siCont. When indicated, cells were ultraviolet-irradiated 15 h before extraction with 2 J m−2 (b) or 10 J m−2 (c). Upper and lower blots were done with the same extracts. (d) Polη downregulation is not at the transcription level. qPCR of POLH and NPM1 mRNA levels from cells pretreated with siRNA against NPM1, normalized to non-targeting siRNA-treated cells. Mean values±s.e.m. of three replicas are presented. (e) Immunoblot of polη extracted from MRC5sv cells pretreated with siRNA against NPM1 and complemented by ectopic expression of an siRNA-resistant NPM1 construct. (f) Polκ, but not polι, is also affected by NPM1 knockdown. Immunoblots of polκ and polι extracted from MRC5sv cells pretreated with siRNA against NPM1, or non-targeting siCont. (g) Accumulation of polη following proteasomal inhibition. MRC5sv cells were treated with non-targeting siRNA or siRNA against NPM1 and then subjected to MG132 for the indicated time periods in order to inhibit proteasomal activity. Tubulin served as a loading control. All blots are representative of three independent experiments. Irad., irradiated.
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f5: NPM1 protects DNA polη from proteasomal degradation.(a) MRC5sv cells stably expressing GFP-POLH were treated with siRNA against NPM1 or non- targeting control siRNA (siCont). GFP-polη levels were quantified by flow cytometry. (b,c) Immunoblot of polη extracted from human XPA cells (b) and MRC5sv cells (c) pretreated with siRNA against NPM1 or non-targeting siCont. When indicated, cells were ultraviolet-irradiated 15 h before extraction with 2 J m−2 (b) or 10 J m−2 (c). Upper and lower blots were done with the same extracts. (d) Polη downregulation is not at the transcription level. qPCR of POLH and NPM1 mRNA levels from cells pretreated with siRNA against NPM1, normalized to non-targeting siRNA-treated cells. Mean values±s.e.m. of three replicas are presented. (e) Immunoblot of polη extracted from MRC5sv cells pretreated with siRNA against NPM1 and complemented by ectopic expression of an siRNA-resistant NPM1 construct. (f) Polκ, but not polι, is also affected by NPM1 knockdown. Immunoblots of polκ and polι extracted from MRC5sv cells pretreated with siRNA against NPM1, or non-targeting siCont. (g) Accumulation of polη following proteasomal inhibition. MRC5sv cells were treated with non-targeting siRNA or siRNA against NPM1 and then subjected to MG132 for the indicated time periods in order to inhibit proteasomal activity. Tubulin served as a loading control. All blots are representative of three independent experiments. Irad., irradiated.

Mentions: To examine whether NPM1 affects polη stability, we measured by flow cytometry the level of ectopically expressed GFP-POLH in MRC5sv cells that stably express this construct. Knocking down NPM1 in these cells resulted in a threefold decrease in the expression of the GFP-polη construct (Fig. 5a, median values). When cells expressing GFP-RAD18, a gene fusion of the main E3 ligase responsible for monoubiquitination of PCNA were analysed, no reduction in RAD18 expression was obtained following knockdown of NPM1 (Supplementary Fig. 6), which is in agreement with the functional PCNA ubiquitination in these cells. Next, we tested whether NPM1 regulates the protein level of endogenous polη. Knocking down the expression of NPM1 in XPA cells caused a significant fourfold decrease in the amount of the NPM1 protein (Fig. 5b, lanes 1 and 2). A similar result was obtained with the MRC5sv cells (3.4-fold decrease; Fig. 5c, lanes 1 and 2). Under these conditions, the amount of polη was significantly reduced both in the XPA cells (3.8-fold decrease; Fig. 5b, lanes 1 and 2) and the repair-proficient MRC5sv cells (2.9-fold decrease, Fig. 5c, lanes 1 and 2), with no significant change in POLH mRNA level, as measured by qPCR (Fig. 5d). A similar effect was observed in ultraviolet-irradiated cells (Fig. 5b lanes 3 and 4, and Fig. 5c lanes 3 and 4, corresponding to XPA and MRC5sv cells, respectively). Importantly, ectopic expression of an siRNA-resistant NPM1 construct rescued the reduction in polη caused by the silencing of endogenous NPM1 (Fig. 5e). Thus, a reduction in the amount of NPM1 causes a decrease in the amount of endogenous polη at the post-transcriptional level. A similar reduction was also observed with polκ, which is another TLS DNA polymerase, but not with polι, the closest homologue of polη in mammalian cells (Fig. 5f). Inhibiting the proteasome using MG132 caused an increase in polη levels in cells pre-treated with siRNA against NPM1, but not in those treated with control siRNA (Fig. 5g). This indicates that NPM1 has an important function in maintaining the stability of polη, and protecting it from proteasomal degradation. The E3 ubiquitin ligases PIRH2 and MDM2 were implicated in the proteasomal degradation of polη. Nevertheless, knocking down each of these enzymes or both, in addition to knocking down NPM1, did not rescue the polη level (Supplementary Fig. 7a,b), suggesting that the degradation of polη upon NPM1 deficiency is not mediated solely by PIRH2 or by MDM2.


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)

NPM1 protects DNA polη from proteasomal degradation.(a) MRC5sv cells stably expressing GFP-POLH were treated with siRNA against NPM1 or non- targeting control siRNA (siCont). GFP-polη levels were quantified by flow cytometry. (b,c) Immunoblot of polη extracted from human XPA cells (b) and MRC5sv cells (c) pretreated with siRNA against NPM1 or non-targeting siCont. When indicated, cells were ultraviolet-irradiated 15 h before extraction with 2 J m−2 (b) or 10 J m−2 (c). Upper and lower blots were done with the same extracts. (d) Polη downregulation is not at the transcription level. qPCR of POLH and NPM1 mRNA levels from cells pretreated with siRNA against NPM1, normalized to non-targeting siRNA-treated cells. Mean values±s.e.m. of three replicas are presented. (e) Immunoblot of polη extracted from MRC5sv cells pretreated with siRNA against NPM1 and complemented by ectopic expression of an siRNA-resistant NPM1 construct. (f) Polκ, but not polι, is also affected by NPM1 knockdown. Immunoblots of polκ and polι extracted from MRC5sv cells pretreated with siRNA against NPM1, or non-targeting siCont. (g) Accumulation of polη following proteasomal inhibition. MRC5sv cells were treated with non-targeting siRNA or siRNA against NPM1 and then subjected to MG132 for the indicated time periods in order to inhibit proteasomal activity. Tubulin served as a loading control. All blots are representative of three independent experiments. Irad., irradiated.
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f5: NPM1 protects DNA polη from proteasomal degradation.(a) MRC5sv cells stably expressing GFP-POLH were treated with siRNA against NPM1 or non- targeting control siRNA (siCont). GFP-polη levels were quantified by flow cytometry. (b,c) Immunoblot of polη extracted from human XPA cells (b) and MRC5sv cells (c) pretreated with siRNA against NPM1 or non-targeting siCont. When indicated, cells were ultraviolet-irradiated 15 h before extraction with 2 J m−2 (b) or 10 J m−2 (c). Upper and lower blots were done with the same extracts. (d) Polη downregulation is not at the transcription level. qPCR of POLH and NPM1 mRNA levels from cells pretreated with siRNA against NPM1, normalized to non-targeting siRNA-treated cells. Mean values±s.e.m. of three replicas are presented. (e) Immunoblot of polη extracted from MRC5sv cells pretreated with siRNA against NPM1 and complemented by ectopic expression of an siRNA-resistant NPM1 construct. (f) Polκ, but not polι, is also affected by NPM1 knockdown. Immunoblots of polκ and polι extracted from MRC5sv cells pretreated with siRNA against NPM1, or non-targeting siCont. (g) Accumulation of polη following proteasomal inhibition. MRC5sv cells were treated with non-targeting siRNA or siRNA against NPM1 and then subjected to MG132 for the indicated time periods in order to inhibit proteasomal activity. Tubulin served as a loading control. All blots are representative of three independent experiments. Irad., irradiated.
Mentions: To examine whether NPM1 affects polη stability, we measured by flow cytometry the level of ectopically expressed GFP-POLH in MRC5sv cells that stably express this construct. Knocking down NPM1 in these cells resulted in a threefold decrease in the expression of the GFP-polη construct (Fig. 5a, median values). When cells expressing GFP-RAD18, a gene fusion of the main E3 ligase responsible for monoubiquitination of PCNA were analysed, no reduction in RAD18 expression was obtained following knockdown of NPM1 (Supplementary Fig. 6), which is in agreement with the functional PCNA ubiquitination in these cells. Next, we tested whether NPM1 regulates the protein level of endogenous polη. Knocking down the expression of NPM1 in XPA cells caused a significant fourfold decrease in the amount of the NPM1 protein (Fig. 5b, lanes 1 and 2). A similar result was obtained with the MRC5sv cells (3.4-fold decrease; Fig. 5c, lanes 1 and 2). Under these conditions, the amount of polη was significantly reduced both in the XPA cells (3.8-fold decrease; Fig. 5b, lanes 1 and 2) and the repair-proficient MRC5sv cells (2.9-fold decrease, Fig. 5c, lanes 1 and 2), with no significant change in POLH mRNA level, as measured by qPCR (Fig. 5d). A similar effect was observed in ultraviolet-irradiated cells (Fig. 5b lanes 3 and 4, and Fig. 5c lanes 3 and 4, corresponding to XPA and MRC5sv cells, respectively). Importantly, ectopic expression of an siRNA-resistant NPM1 construct rescued the reduction in polη caused by the silencing of endogenous NPM1 (Fig. 5e). Thus, a reduction in the amount of NPM1 causes a decrease in the amount of endogenous polη at the post-transcriptional level. A similar reduction was also observed with polκ, which is another TLS DNA polymerase, but not with polι, the closest homologue of polη in mammalian cells (Fig. 5f). Inhibiting the proteasome using MG132 caused an increase in polη levels in cells pre-treated with siRNA against NPM1, but not in those treated with control siRNA (Fig. 5g). This indicates that NPM1 has an important function in maintaining the stability of polη, and protecting it from proteasomal degradation. The E3 ubiquitin ligases PIRH2 and MDM2 were implicated in the proteasomal degradation of polη. Nevertheless, knocking down each of these enzymes or both, in addition to knocking down NPM1, did not rescue the polη level (Supplementary Fig. 7a,b), suggesting that the degradation of polη upon NPM1 deficiency is not mediated solely by PIRH2 or by MDM2.

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