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The CDC13-STN1-TEN1 complex stimulates Pol α activity by promoting RNA priming and primase-to-polymerase switch.

Lue NF, Chan J, Wright WE, Hurwitz J - Nat Commun (2014)

Bottom Line: While CST does not enhance isolated DNA polymerase activity, it substantially augments both primase activity and primase-to-polymerase switching.Both the N-terminal OB fold and the C-terminal winged-helix domains of Stn1 can bind to the Pol12 subunit of the PP complex and stimulate PP activity.Our findings provide mechanistic insights on a well-conserved pathway of PP regulation that is critical for genome stability.

View Article: PubMed Central - PubMed

Affiliation: W. R. Hearst Microbiology Research Center, Department of Microbiology &Immunology, Weill Medical College of Cornell University, New York, New York 10065, USA.

ABSTRACT
Emerging evidence suggests that Cdc13-Stn1-Ten1 (CST), an RPA-like ssDNA-binding complex, may regulate primase-Pol α (PP) activity at telomeres constitutively, and at other genomic locations under conditions of replication stress. Here we examine the mechanisms of PP stimulation by CST using purified complexes derived from Candida glabrata. While CST does not enhance isolated DNA polymerase activity, it substantially augments both primase activity and primase-to-polymerase switching. CST also simultaneously shortens the RNA and lengthens the DNA in the chimeric products. Stn1, the most conserved subunit of CST, is alone capable of PP stimulation. Both the N-terminal OB fold and the C-terminal winged-helix domains of Stn1 can bind to the Pol12 subunit of the PP complex and stimulate PP activity. Our findings provide mechanistic insights on a well-conserved pathway of PP regulation that is critical for genome stability.

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Stimulation of PP activity by CST, Stn1 and Stn1 variants(a) (Left) The effects of CST complex and individual subunits (450 nM) on PP activity (2 nM) were analyzed in the coupled primase-polymerase assays using the poly-dT template. (Right) The effects of CST complex and individual subunits (450 nM) on PP activity (2 nM) were analyzed in the coupled primase-polymerase assays using the CgG4 template. (b) The effects of Full length Stn1 and the N- and C-terminus of Stn1 (1 µM) on PP (2 nM) activity were analyzed in the coupled primase-polymerase assays using poly-dT template.(c) PP (1 nM) activity was assayed using poly-dT and varying concentrations (75, 150 and 600 nM) of Stn1, Stn1N, and Stn1C. (d) The stimulatory effects (averages S.D.) of varying concentrations of CST, Stn1, Stn1N and Stn1C from three independent experiments were quantified and plotted. (e) The stimulatory effects of WH1 and WH2 motifs of Stn1C (at 100 and 300 nM concentrations) were analyzed in the coupled primase-polymerase assays using the poly-dT template. The assays were from the same gel with several irrelevant lanes cropped out.
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Figure 3: Stimulation of PP activity by CST, Stn1 and Stn1 variants(a) (Left) The effects of CST complex and individual subunits (450 nM) on PP activity (2 nM) were analyzed in the coupled primase-polymerase assays using the poly-dT template. (Right) The effects of CST complex and individual subunits (450 nM) on PP activity (2 nM) were analyzed in the coupled primase-polymerase assays using the CgG4 template. (b) The effects of Full length Stn1 and the N- and C-terminus of Stn1 (1 µM) on PP (2 nM) activity were analyzed in the coupled primase-polymerase assays using poly-dT template.(c) PP (1 nM) activity was assayed using poly-dT and varying concentrations (75, 150 and 600 nM) of Stn1, Stn1N, and Stn1C. (d) The stimulatory effects (averages S.D.) of varying concentrations of CST, Stn1, Stn1N and Stn1C from three independent experiments were quantified and plotted. (e) The stimulatory effects of WH1 and WH2 motifs of Stn1C (at 100 and 300 nM concentrations) were analyzed in the coupled primase-polymerase assays using the poly-dT template. The assays were from the same gel with several irrelevant lanes cropped out.

Mentions: To determine the subunit(s) of the CST complex responsible for PP stimulation, we prepared and tested isolated subunits. Each subunit was expressed in E. coli, purified by affinity chromatography (Supplementary Fig. 1a), and found to be free of contaminating polymerase activity. Notably, neither Cdc13 nor Ten1 was capable of stimulating PP. Because we had previously shown that recombinant Cdc13 alone is active in DNA binding, its inability to stimulate PP cannot be attributed to gross misfolding41. In contrast to Cdc13 and Ten1, Stn1 alone was nearly active as the intact CST complex in PP stimulation (Fig. 3a). The effect of Stn1 can be observed on both the Poly-dT and C. glabrata G-tail templates (Fig. 3a, left and right panels). Stn1 contains an N-terminal OB fold domain that is implicated in DNA binding, and a C-terminal duplicated winged-helix domain that is proposed to contact other proteins36,37,39,41. We purified these two domains separately (Supplementary Fig. 1a) and found that each was competent in PP stimulation (Fig. 3b). All the Stn1 variants (i.e., full length, N-terminus and C-terminus) caused the characteristic lengthening of products described earlier for the intact CST complex, suggesting that their mechanisms of stimulation are likely to be similar. Titration of CST and Stn1 variants in the Poly-dT assays indicates that CST, Stn1, and Stn1N have comparable activities, while Stn1C is somewhat less active in PP stimulation (Fig. 3c, d). Thus, the PP-regulatory function of CST appears to reside largely in the Stn1 subunit, which possesses at least two determinants for promoting PP activity.


The CDC13-STN1-TEN1 complex stimulates Pol α activity by promoting RNA priming and primase-to-polymerase switch.

Lue NF, Chan J, Wright WE, Hurwitz J - Nat Commun (2014)

Stimulation of PP activity by CST, Stn1 and Stn1 variants(a) (Left) The effects of CST complex and individual subunits (450 nM) on PP activity (2 nM) were analyzed in the coupled primase-polymerase assays using the poly-dT template. (Right) The effects of CST complex and individual subunits (450 nM) on PP activity (2 nM) were analyzed in the coupled primase-polymerase assays using the CgG4 template. (b) The effects of Full length Stn1 and the N- and C-terminus of Stn1 (1 µM) on PP (2 nM) activity were analyzed in the coupled primase-polymerase assays using poly-dT template.(c) PP (1 nM) activity was assayed using poly-dT and varying concentrations (75, 150 and 600 nM) of Stn1, Stn1N, and Stn1C. (d) The stimulatory effects (averages S.D.) of varying concentrations of CST, Stn1, Stn1N and Stn1C from three independent experiments were quantified and plotted. (e) The stimulatory effects of WH1 and WH2 motifs of Stn1C (at 100 and 300 nM concentrations) were analyzed in the coupled primase-polymerase assays using the poly-dT template. The assays were from the same gel with several irrelevant lanes cropped out.
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Related In: Results  -  Collection

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Figure 3: Stimulation of PP activity by CST, Stn1 and Stn1 variants(a) (Left) The effects of CST complex and individual subunits (450 nM) on PP activity (2 nM) were analyzed in the coupled primase-polymerase assays using the poly-dT template. (Right) The effects of CST complex and individual subunits (450 nM) on PP activity (2 nM) were analyzed in the coupled primase-polymerase assays using the CgG4 template. (b) The effects of Full length Stn1 and the N- and C-terminus of Stn1 (1 µM) on PP (2 nM) activity were analyzed in the coupled primase-polymerase assays using poly-dT template.(c) PP (1 nM) activity was assayed using poly-dT and varying concentrations (75, 150 and 600 nM) of Stn1, Stn1N, and Stn1C. (d) The stimulatory effects (averages S.D.) of varying concentrations of CST, Stn1, Stn1N and Stn1C from three independent experiments were quantified and plotted. (e) The stimulatory effects of WH1 and WH2 motifs of Stn1C (at 100 and 300 nM concentrations) were analyzed in the coupled primase-polymerase assays using the poly-dT template. The assays were from the same gel with several irrelevant lanes cropped out.
Mentions: To determine the subunit(s) of the CST complex responsible for PP stimulation, we prepared and tested isolated subunits. Each subunit was expressed in E. coli, purified by affinity chromatography (Supplementary Fig. 1a), and found to be free of contaminating polymerase activity. Notably, neither Cdc13 nor Ten1 was capable of stimulating PP. Because we had previously shown that recombinant Cdc13 alone is active in DNA binding, its inability to stimulate PP cannot be attributed to gross misfolding41. In contrast to Cdc13 and Ten1, Stn1 alone was nearly active as the intact CST complex in PP stimulation (Fig. 3a). The effect of Stn1 can be observed on both the Poly-dT and C. glabrata G-tail templates (Fig. 3a, left and right panels). Stn1 contains an N-terminal OB fold domain that is implicated in DNA binding, and a C-terminal duplicated winged-helix domain that is proposed to contact other proteins36,37,39,41. We purified these two domains separately (Supplementary Fig. 1a) and found that each was competent in PP stimulation (Fig. 3b). All the Stn1 variants (i.e., full length, N-terminus and C-terminus) caused the characteristic lengthening of products described earlier for the intact CST complex, suggesting that their mechanisms of stimulation are likely to be similar. Titration of CST and Stn1 variants in the Poly-dT assays indicates that CST, Stn1, and Stn1N have comparable activities, while Stn1C is somewhat less active in PP stimulation (Fig. 3c, d). Thus, the PP-regulatory function of CST appears to reside largely in the Stn1 subunit, which possesses at least two determinants for promoting PP activity.

Bottom Line: While CST does not enhance isolated DNA polymerase activity, it substantially augments both primase activity and primase-to-polymerase switching.Both the N-terminal OB fold and the C-terminal winged-helix domains of Stn1 can bind to the Pol12 subunit of the PP complex and stimulate PP activity.Our findings provide mechanistic insights on a well-conserved pathway of PP regulation that is critical for genome stability.

View Article: PubMed Central - PubMed

Affiliation: W. R. Hearst Microbiology Research Center, Department of Microbiology &Immunology, Weill Medical College of Cornell University, New York, New York 10065, USA.

ABSTRACT
Emerging evidence suggests that Cdc13-Stn1-Ten1 (CST), an RPA-like ssDNA-binding complex, may regulate primase-Pol α (PP) activity at telomeres constitutively, and at other genomic locations under conditions of replication stress. Here we examine the mechanisms of PP stimulation by CST using purified complexes derived from Candida glabrata. While CST does not enhance isolated DNA polymerase activity, it substantially augments both primase activity and primase-to-polymerase switching. CST also simultaneously shortens the RNA and lengthens the DNA in the chimeric products. Stn1, the most conserved subunit of CST, is alone capable of PP stimulation. Both the N-terminal OB fold and the C-terminal winged-helix domains of Stn1 can bind to the Pol12 subunit of the PP complex and stimulate PP activity. Our findings provide mechanistic insights on a well-conserved pathway of PP regulation that is critical for genome stability.

Show MeSH