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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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Secondary functional TLS screen.(a,b) The plasmids used for the secondary TLS screen: A Fluc-expressing gapped plasmid carrying a site-specific DNA lesion on the non-transcribed strand and a gap on the transcribed strand (a) and a control Rluc-expressing gapped plasmid without a lesion (b). (c) An outline of the secondary TLS screen. In brief, XPA cells were transfected with the siRNA on day 0 and with the reporter plasmids on day 2. Fluc and Rluc signals were measured on day 3. (d,e) Histograms describing TLS FC effects caused by the siRNAs (median values over four replicas that were preformed on different days). Dashed red lines denote the distribution borders of negative control samples. Data from the TT 6-4 PP TLS screen is presented in d, and that from the TT CPD TLS screen in e. (f,g) Luminescence values of the four biological replicas plotted against each other from the TT 6-4 PP screen (f), and the TT CPD screen (g). (h) Enrichment of TLS, DNA repair and DNA-damage response pathways within the screen hits. The dashed red line corresponds to a hypergeometric test P value 0.05. See also Supplementary Fig. 2, Supplementary Data 1 and Supplementary Tables 1 and 2.
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f2: Secondary functional TLS screen.(a,b) The plasmids used for the secondary TLS screen: A Fluc-expressing gapped plasmid carrying a site-specific DNA lesion on the non-transcribed strand and a gap on the transcribed strand (a) and a control Rluc-expressing gapped plasmid without a lesion (b). (c) An outline of the secondary TLS screen. In brief, XPA cells were transfected with the siRNA on day 0 and with the reporter plasmids on day 2. Fluc and Rluc signals were measured on day 3. (d,e) Histograms describing TLS FC effects caused by the siRNAs (median values over four replicas that were preformed on different days). Dashed red lines denote the distribution borders of negative control samples. Data from the TT 6-4 PP TLS screen is presented in d, and that from the TT CPD TLS screen in e. (f,g) Luminescence values of the four biological replicas plotted against each other from the TT 6-4 PP screen (f), and the TT CPD screen (g). (h) Enrichment of TLS, DNA repair and DNA-damage response pathways within the screen hits. The dashed red line corresponds to a hypergeometric test P value 0.05. See also Supplementary Fig. 2, Supplementary Data 1 and Supplementary Tables 1 and 2.

Mentions: The second screening stage was performed using a newly developed high-throughput TLS assay. It is a modification of an assay based on plasmids containing a site-specific DNA lesion opposite a gap, previously successfully used to study TLS173238. In the new assay, the gap lesion is positioned between a cytomegalovirus (CMV) promoter and a firefly luciferase (Fluc) reporter gene (Fig. 2a). The presence of a gap in the transcribed strand does not allow expression of the Fluc gene, unless the missing segment of the coding strand is synthesized by TLS. A lesion-free gapped plasmid expressing Renilla luciferase (Rluc) served to normalize for transfection and gap-filling efficiencies (Fig. 2b). Of note, the product of a successful TLS event in the Fluc system still carries the lesion on the non-transcribed strand, which might interfere with Fluc expression. To test this possibility, we constructed the expected TLS product, namely a fully double-stranded plasmid with the lesion on the non-transcribed strand, and found that its Fluc expression in XPA cells was essentially identical to that obtained with a control plasmid without the lesion (Supplementary Fig. 2a).


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)

Secondary functional TLS screen.(a,b) The plasmids used for the secondary TLS screen: A Fluc-expressing gapped plasmid carrying a site-specific DNA lesion on the non-transcribed strand and a gap on the transcribed strand (a) and a control Rluc-expressing gapped plasmid without a lesion (b). (c) An outline of the secondary TLS screen. In brief, XPA cells were transfected with the siRNA on day 0 and with the reporter plasmids on day 2. Fluc and Rluc signals were measured on day 3. (d,e) Histograms describing TLS FC effects caused by the siRNAs (median values over four replicas that were preformed on different days). Dashed red lines denote the distribution borders of negative control samples. Data from the TT 6-4 PP TLS screen is presented in d, and that from the TT CPD TLS screen in e. (f,g) Luminescence values of the four biological replicas plotted against each other from the TT 6-4 PP screen (f), and the TT CPD screen (g). (h) Enrichment of TLS, DNA repair and DNA-damage response pathways within the screen hits. The dashed red line corresponds to a hypergeometric test P value 0.05. See also Supplementary Fig. 2, Supplementary Data 1 and Supplementary Tables 1 and 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Secondary functional TLS screen.(a,b) The plasmids used for the secondary TLS screen: A Fluc-expressing gapped plasmid carrying a site-specific DNA lesion on the non-transcribed strand and a gap on the transcribed strand (a) and a control Rluc-expressing gapped plasmid without a lesion (b). (c) An outline of the secondary TLS screen. In brief, XPA cells were transfected with the siRNA on day 0 and with the reporter plasmids on day 2. Fluc and Rluc signals were measured on day 3. (d,e) Histograms describing TLS FC effects caused by the siRNAs (median values over four replicas that were preformed on different days). Dashed red lines denote the distribution borders of negative control samples. Data from the TT 6-4 PP TLS screen is presented in d, and that from the TT CPD TLS screen in e. (f,g) Luminescence values of the four biological replicas plotted against each other from the TT 6-4 PP screen (f), and the TT CPD screen (g). (h) Enrichment of TLS, DNA repair and DNA-damage response pathways within the screen hits. The dashed red line corresponds to a hypergeometric test P value 0.05. See also Supplementary Fig. 2, Supplementary Data 1 and Supplementary Tables 1 and 2.
Mentions: The second screening stage was performed using a newly developed high-throughput TLS assay. It is a modification of an assay based on plasmids containing a site-specific DNA lesion opposite a gap, previously successfully used to study TLS173238. In the new assay, the gap lesion is positioned between a cytomegalovirus (CMV) promoter and a firefly luciferase (Fluc) reporter gene (Fig. 2a). The presence of a gap in the transcribed strand does not allow expression of the Fluc gene, unless the missing segment of the coding strand is synthesized by TLS. A lesion-free gapped plasmid expressing Renilla luciferase (Rluc) served to normalize for transfection and gap-filling efficiencies (Fig. 2b). Of note, the product of a successful TLS event in the Fluc system still carries the lesion on the non-transcribed strand, which might interfere with Fluc expression. To test this possibility, we constructed the expected TLS product, namely a fully double-stranded plasmid with the lesion on the non-transcribed strand, and found that its Fluc expression in XPA cells was essentially identical to that obtained with a control plasmid without the lesion (Supplementary Fig. 2a).

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