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DNA damage signalling prevents deleterious telomere addition at DNA breaks.

Makovets S, Blackburn EH - Nat. Cell Biol. (2009)

Bottom Line: Here, we report that telomerase action is regulated as a part of the cellular response to DNA double-strand breaks (DSBs).Using a separation of function PIF1 mutation, we show that this phosphorylation is specifically required for the Pif1-mediated telomerase inhibition that takes place at DNA breaks, but not for that at telomeres.Hence DNA damage signalling down-modulates telomerase action at DNA breaks through Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.

ABSTRACT
The response to DNA damage involves regulation of several essential processes to maximize the accuracy of DNA damage repair and cell survival. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage to increase the accuracy of repair. Here, we report that telomerase action is regulated as a part of the cellular response to DNA double-strand breaks (DSBs). Using yeast, we show that the main ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. After DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Using a separation of function PIF1 mutation, we show that this phosphorylation is specifically required for the Pif1-mediated telomerase inhibition that takes place at DNA breaks, but not for that at telomeres. Hence DNA damage signalling down-modulates telomerase action at DNA breaks through Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a new regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity.

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pif1-4A is a separation of function phospho-site mutant, defective in telomerase inhibition specifically at DSBs and not telomeres. a, Mutagenesis based scanning of nPif1 for potential phosphorylation loci involved in its function during DNA damage response. Schematic of the multiple S/T→A substitutions constructed in different nPif1 regions (See Methods for details) and summary of mutant phenotypes (on the right, data presented in Fig. S2). b, Telomere length analysis in PIF1, pif1-m2, pif1-4D, and pif1-4A cells. c, Frequency of 5-FOAR colony formation in PIF1, pif1-m2, pif1-4A, and pif1-4D upon DSB induction. Error bars represent average ± s.d. from four independent measurements for each strain, except pif1-4D (three measurements). Note that the majority of 5-FOAR colonies from PIF1 cells do not represent de novo telomere addition events whereas the majority of the same class of clones from pif1-m2 and pif1-4A do (see the numbers under the graph and Fig. S3). d, Analysis of nPif1-4myc and nPif1-4A-4myc localization to a galactose inducible DSB by chromatin immunoprecipitation. Error bars show average ± s.d. from four independent experiments. e, TLSSAES is phosphorylated in response to a single DSB. Note that the anti-P-Pif1 antibody has weak cross-reactivity with another DNA damage induced phosphorylation site on Pif1 as seen in lane 6 (pif1-4A in galactose). However, this does not affect the conclusiveness of the data as there is a significant signal difference between PIF1 and pif1-4A (compare lanes 4 and 6). The same applies to panels f and g. f, TLSSAES phosphorylation in response to a single DSB requires MEC1, RAD53, and DUN1. g, TLSSAES is phosphorylated in response to DSBs but not in response to nocodazole-induced G2 arrest. h, TLSSAES is phosphorylated in response to DSBs (DSB and Phleomycin, Phl) but not stalled replication forks (hydroxyurea, HU). DNA damage was induced as in Fig. 1. In the panels e–h, samples of immunoprecipitated nPif1-4myc were analyzed by western blotting using an affinity purified rabbit polyclonal antibody raised against VIDFYL(pT)LS(pS)AE (anti-P-Pif1, upper blot on each panel) and then re-probed with anti-myc antibody (lower blot).
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Figure 2: pif1-4A is a separation of function phospho-site mutant, defective in telomerase inhibition specifically at DSBs and not telomeres. a, Mutagenesis based scanning of nPif1 for potential phosphorylation loci involved in its function during DNA damage response. Schematic of the multiple S/T→A substitutions constructed in different nPif1 regions (See Methods for details) and summary of mutant phenotypes (on the right, data presented in Fig. S2). b, Telomere length analysis in PIF1, pif1-m2, pif1-4D, and pif1-4A cells. c, Frequency of 5-FOAR colony formation in PIF1, pif1-m2, pif1-4A, and pif1-4D upon DSB induction. Error bars represent average ± s.d. from four independent measurements for each strain, except pif1-4D (three measurements). Note that the majority of 5-FOAR colonies from PIF1 cells do not represent de novo telomere addition events whereas the majority of the same class of clones from pif1-m2 and pif1-4A do (see the numbers under the graph and Fig. S3). d, Analysis of nPif1-4myc and nPif1-4A-4myc localization to a galactose inducible DSB by chromatin immunoprecipitation. Error bars show average ± s.d. from four independent experiments. e, TLSSAES is phosphorylated in response to a single DSB. Note that the anti-P-Pif1 antibody has weak cross-reactivity with another DNA damage induced phosphorylation site on Pif1 as seen in lane 6 (pif1-4A in galactose). However, this does not affect the conclusiveness of the data as there is a significant signal difference between PIF1 and pif1-4A (compare lanes 4 and 6). The same applies to panels f and g. f, TLSSAES phosphorylation in response to a single DSB requires MEC1, RAD53, and DUN1. g, TLSSAES is phosphorylated in response to DSBs but not in response to nocodazole-induced G2 arrest. h, TLSSAES is phosphorylated in response to DSBs (DSB and Phleomycin, Phl) but not stalled replication forks (hydroxyurea, HU). DNA damage was induced as in Fig. 1. In the panels e–h, samples of immunoprecipitated nPif1-4myc were analyzed by western blotting using an affinity purified rabbit polyclonal antibody raised against VIDFYL(pT)LS(pS)AE (anti-P-Pif1, upper blot on each panel) and then re-probed with anti-myc antibody (lower blot).

Mentions: In the absence of nPif1, cells have longer telomeres and telomerase heals DSBs inappropriately by adding a new telomere ~200 times more frequently than in PIF1 cells 6. We searched for a Pif1 locus, potentially a phosphorylation site, important for the telomerase-inhibitory action of Pif1 that specifically occurs during a DNA damage response, i.e. at DSBs. S. cerevisiae Pif1 contains two regions - helicase motifs I–IV and V–VI - homologous to other helicases 13. The rest of the Pif1 protein, i.e. the middle part between the motif-containing regions as well as the N- and the C-terminal portions, have no obvious homologies, and we tested their involvement in the regulation of Pif1 by DNA damage signalling (Fig. 2a). Protein phosphorylation prediction program NetPhos 2.0 (www.cbs.dtu.dk) was used to scan Pif1 for potential phosphorylation at serine and threonine residues. Those residues with a prediction value above 0.25 in either the N-terminal or in the middle regions were mutated to non-phosphorylatable alanines to generate PIF1 alleles with multiple substitutions, pif1-N-18A and pif1-M-11A respectively (Fig. 2a). In the C-terminus, all serines and threonines from T763 to the end were replaced with alanines to generate pif1-C-18A.


DNA damage signalling prevents deleterious telomere addition at DNA breaks.

Makovets S, Blackburn EH - Nat. Cell Biol. (2009)

pif1-4A is a separation of function phospho-site mutant, defective in telomerase inhibition specifically at DSBs and not telomeres. a, Mutagenesis based scanning of nPif1 for potential phosphorylation loci involved in its function during DNA damage response. Schematic of the multiple S/T→A substitutions constructed in different nPif1 regions (See Methods for details) and summary of mutant phenotypes (on the right, data presented in Fig. S2). b, Telomere length analysis in PIF1, pif1-m2, pif1-4D, and pif1-4A cells. c, Frequency of 5-FOAR colony formation in PIF1, pif1-m2, pif1-4A, and pif1-4D upon DSB induction. Error bars represent average ± s.d. from four independent measurements for each strain, except pif1-4D (three measurements). Note that the majority of 5-FOAR colonies from PIF1 cells do not represent de novo telomere addition events whereas the majority of the same class of clones from pif1-m2 and pif1-4A do (see the numbers under the graph and Fig. S3). d, Analysis of nPif1-4myc and nPif1-4A-4myc localization to a galactose inducible DSB by chromatin immunoprecipitation. Error bars show average ± s.d. from four independent experiments. e, TLSSAES is phosphorylated in response to a single DSB. Note that the anti-P-Pif1 antibody has weak cross-reactivity with another DNA damage induced phosphorylation site on Pif1 as seen in lane 6 (pif1-4A in galactose). However, this does not affect the conclusiveness of the data as there is a significant signal difference between PIF1 and pif1-4A (compare lanes 4 and 6). The same applies to panels f and g. f, TLSSAES phosphorylation in response to a single DSB requires MEC1, RAD53, and DUN1. g, TLSSAES is phosphorylated in response to DSBs but not in response to nocodazole-induced G2 arrest. h, TLSSAES is phosphorylated in response to DSBs (DSB and Phleomycin, Phl) but not stalled replication forks (hydroxyurea, HU). DNA damage was induced as in Fig. 1. In the panels e–h, samples of immunoprecipitated nPif1-4myc were analyzed by western blotting using an affinity purified rabbit polyclonal antibody raised against VIDFYL(pT)LS(pS)AE (anti-P-Pif1, upper blot on each panel) and then re-probed with anti-myc antibody (lower blot).
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Show All Figures
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Figure 2: pif1-4A is a separation of function phospho-site mutant, defective in telomerase inhibition specifically at DSBs and not telomeres. a, Mutagenesis based scanning of nPif1 for potential phosphorylation loci involved in its function during DNA damage response. Schematic of the multiple S/T→A substitutions constructed in different nPif1 regions (See Methods for details) and summary of mutant phenotypes (on the right, data presented in Fig. S2). b, Telomere length analysis in PIF1, pif1-m2, pif1-4D, and pif1-4A cells. c, Frequency of 5-FOAR colony formation in PIF1, pif1-m2, pif1-4A, and pif1-4D upon DSB induction. Error bars represent average ± s.d. from four independent measurements for each strain, except pif1-4D (three measurements). Note that the majority of 5-FOAR colonies from PIF1 cells do not represent de novo telomere addition events whereas the majority of the same class of clones from pif1-m2 and pif1-4A do (see the numbers under the graph and Fig. S3). d, Analysis of nPif1-4myc and nPif1-4A-4myc localization to a galactose inducible DSB by chromatin immunoprecipitation. Error bars show average ± s.d. from four independent experiments. e, TLSSAES is phosphorylated in response to a single DSB. Note that the anti-P-Pif1 antibody has weak cross-reactivity with another DNA damage induced phosphorylation site on Pif1 as seen in lane 6 (pif1-4A in galactose). However, this does not affect the conclusiveness of the data as there is a significant signal difference between PIF1 and pif1-4A (compare lanes 4 and 6). The same applies to panels f and g. f, TLSSAES phosphorylation in response to a single DSB requires MEC1, RAD53, and DUN1. g, TLSSAES is phosphorylated in response to DSBs but not in response to nocodazole-induced G2 arrest. h, TLSSAES is phosphorylated in response to DSBs (DSB and Phleomycin, Phl) but not stalled replication forks (hydroxyurea, HU). DNA damage was induced as in Fig. 1. In the panels e–h, samples of immunoprecipitated nPif1-4myc were analyzed by western blotting using an affinity purified rabbit polyclonal antibody raised against VIDFYL(pT)LS(pS)AE (anti-P-Pif1, upper blot on each panel) and then re-probed with anti-myc antibody (lower blot).
Mentions: In the absence of nPif1, cells have longer telomeres and telomerase heals DSBs inappropriately by adding a new telomere ~200 times more frequently than in PIF1 cells 6. We searched for a Pif1 locus, potentially a phosphorylation site, important for the telomerase-inhibitory action of Pif1 that specifically occurs during a DNA damage response, i.e. at DSBs. S. cerevisiae Pif1 contains two regions - helicase motifs I–IV and V–VI - homologous to other helicases 13. The rest of the Pif1 protein, i.e. the middle part between the motif-containing regions as well as the N- and the C-terminal portions, have no obvious homologies, and we tested their involvement in the regulation of Pif1 by DNA damage signalling (Fig. 2a). Protein phosphorylation prediction program NetPhos 2.0 (www.cbs.dtu.dk) was used to scan Pif1 for potential phosphorylation at serine and threonine residues. Those residues with a prediction value above 0.25 in either the N-terminal or in the middle regions were mutated to non-phosphorylatable alanines to generate PIF1 alleles with multiple substitutions, pif1-N-18A and pif1-M-11A respectively (Fig. 2a). In the C-terminus, all serines and threonines from T763 to the end were replaced with alanines to generate pif1-C-18A.

Bottom Line: Here, we report that telomerase action is regulated as a part of the cellular response to DNA double-strand breaks (DSBs).Using a separation of function PIF1 mutation, we show that this phosphorylation is specifically required for the Pif1-mediated telomerase inhibition that takes place at DNA breaks, but not for that at telomeres.Hence DNA damage signalling down-modulates telomerase action at DNA breaks through Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.

ABSTRACT
The response to DNA damage involves regulation of several essential processes to maximize the accuracy of DNA damage repair and cell survival. Telomerase has the potential to interfere with repair by inappropriately adding telomeres to DNA breaks. It was unknown whether cells modulate telomerase in response to DNA damage to increase the accuracy of repair. Here, we report that telomerase action is regulated as a part of the cellular response to DNA double-strand breaks (DSBs). Using yeast, we show that the main ATR/Mec1 DNA damage signalling pathway regulates telomerase action at DSBs. After DNA damage, MEC1-RAD53-DUN1-dependent phosphorylation of the telomerase inhibitor Pif1 occurs. Using a separation of function PIF1 mutation, we show that this phosphorylation is specifically required for the Pif1-mediated telomerase inhibition that takes place at DNA breaks, but not for that at telomeres. Hence DNA damage signalling down-modulates telomerase action at DNA breaks through Pif1 phosphorylation, thus preventing aberrant healing of broken DNA ends by telomerase. These findings uncover a new regulatory mechanism that coordinates competing DNA end-processing activities and thereby promotes DNA repair accuracy and genome integrity.

Show MeSH
Related in: MedlinePlus