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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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Related in: MedlinePlus

CST promotes RNA priming and the primase-to-polymerase transition(a) The primase activity of the PP complex (1 nM) was analyzed at the indicated ATP concentrations in the absence or presence of CST (100 nM). Poly-dT was included at 300 nM, and P32-ATP was included at 12 µCi nmole−1 and 2.4 µCi nmole−1 for the 20 µM and 400 µM ATP reactions, respectively. Size standards in this and several other assays include both ssDNA (19, 37, 55 and 97 nt) and ssRNA (rA10). Results (averages S.D.) from three independent assays were quantified and plotted. (b) The two steps of the coupled “primase-Klenow” assay are illustrated on the left, and total32P incorporations plotted on the right. As controls, the priming step of the reactions was performed using no ribonucleotides or GTP only. Data (averages S.D.) are from three independent sets of assays.(c) The DNA polymerase activity of PP (1 nM) was analyzed in the absence or presence of CST (100 nM) using as substrates “pre-primed” poly-dT/rA10 (150 nM and 450 nM). (d) The DNA polymerase activity of PP (1 nM) was analyzed in the absence or presence of CST (150 nM) using as substrates the poly-dT/oligo-rA synthesized by PP in a priming reaction. (e) The primase-to-polymerase switch efficiency of PP (2 nM) was analyzed in the absence or presence of CST (100 and 200 nM) at the indicated ATP concentrations. P32-ATP was included at 24 µCi nmole−1 and 1.2 µCi nmole−1 for the 20 µM and 400 µM ATP reactions, respectively. (Because the Km of the primase for ribonucleotides is ~150 µM8, higher specific activity of P32-ATP is needed to generate robust signals in the 20 µM ATP reactions). (f) The fractions of extendable RNA primers (i.e., 7–10 nt long) that were not lengthened by the polymerase into RNA-DNA chimeras in assays such as those shown in D were quantified and plotted. Data (averages S.D.) are from three independent sets of assays. (g) The total numbers of RNA and RNA-DNA products in assays such as those shown in D were calculated by summing the normalized intensities (normalized against the number of labeled rA in each product) and plotted. (For the RNA-DNA oligomer, we assume an average RNA length of 9.) Data (averages S.D.) are from three independent sets of assays.
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Figure 2: CST promotes RNA priming and the primase-to-polymerase transition(a) The primase activity of the PP complex (1 nM) was analyzed at the indicated ATP concentrations in the absence or presence of CST (100 nM). Poly-dT was included at 300 nM, and P32-ATP was included at 12 µCi nmole−1 and 2.4 µCi nmole−1 for the 20 µM and 400 µM ATP reactions, respectively. Size standards in this and several other assays include both ssDNA (19, 37, 55 and 97 nt) and ssRNA (rA10). Results (averages S.D.) from three independent assays were quantified and plotted. (b) The two steps of the coupled “primase-Klenow” assay are illustrated on the left, and total32P incorporations plotted on the right. As controls, the priming step of the reactions was performed using no ribonucleotides or GTP only. Data (averages S.D.) are from three independent sets of assays.(c) The DNA polymerase activity of PP (1 nM) was analyzed in the absence or presence of CST (100 nM) using as substrates “pre-primed” poly-dT/rA10 (150 nM and 450 nM). (d) The DNA polymerase activity of PP (1 nM) was analyzed in the absence or presence of CST (150 nM) using as substrates the poly-dT/oligo-rA synthesized by PP in a priming reaction. (e) The primase-to-polymerase switch efficiency of PP (2 nM) was analyzed in the absence or presence of CST (100 and 200 nM) at the indicated ATP concentrations. P32-ATP was included at 24 µCi nmole−1 and 1.2 µCi nmole−1 for the 20 µM and 400 µM ATP reactions, respectively. (Because the Km of the primase for ribonucleotides is ~150 µM8, higher specific activity of P32-ATP is needed to generate robust signals in the 20 µM ATP reactions). (f) The fractions of extendable RNA primers (i.e., 7–10 nt long) that were not lengthened by the polymerase into RNA-DNA chimeras in assays such as those shown in D were quantified and plotted. Data (averages S.D.) are from three independent sets of assays. (g) The total numbers of RNA and RNA-DNA products in assays such as those shown in D were calculated by summing the normalized intensities (normalized against the number of labeled rA in each product) and plotted. (For the RNA-DNA oligomer, we assume an average RNA length of 9.) Data (averages S.D.) are from three independent sets of assays.

Mentions: A priori, the stimulatory effect of CST on total product synthesis can be due to an enhancement of the primase or DNA polymerase reaction, or the “coupling” between the two active sites. To distinguish between these possibilities, we first analyzed the effect of CST in separate primase and DNA polymerase reactions. For the primase-only assay, PP was incubated with poly-dT in the presence of labeled ATP as the sole nucleotide. Previous studies indicate that eukaryotic primase preferentially synthesizes unit-length primers (7–10 nt) or multiples thereof (i.e., multiples of 7–10 nt) in the absence of dNTP. Indeed, we observed both unit-length primers and dimers in our assays (Fig. 2a). More interestingly, the effect of CST in this assay was ATP concentration-dependent: CST stimulated primer synthesis by 2–3 fold in 20 µM ATP, but had much less effect in 400 µM ATP (Fig. 2a). As a complementary test, we performed a standard primase assay, which combines primer synthesis (using unlabeled 3 mM ATP) with extension by Klenow (using labeled dATP)2. Because Klenow was included in excess, the dATP incorporation was proportional to the RNA primers produced in the reaction. Using this assay, we found little stimulation of primase activity by CST (Fig. 2b). Thus, CST preferentially enhances primase activity at low ATP concentrations. We then performed polymerase-only assays, for which we used “pre-primed” Poly-dT/rA10 template and dATP as the substrate (Fig. 2c). No enhancement of DNA synthesis was observed in the presence of CST, indicating that the complex does not stimulate the DNA polymerase activity. We reasoned that the DNA template/RNA primer generated by primase might behave differently from the synthetic Poly-dT/rA10 substrate, and hence performed an alternative assay. In this assay, we first allowed PP to synthesize unlabeled oligo-rA using ATP as the sole nucleotide substrate. Upon completion of RNA synthesis, free ATP was removed from the reaction, and the resulting Poly-dT/oligo-rA mixture used as the substrate for DNA synthesis by PP in the absence or presence of CST (Fig. 2d). CST again had no effect on DNA synthesis, indicating that it does not stimulate “isolated” DNA polymerase α 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)

CST promotes RNA priming and the primase-to-polymerase transition(a) The primase activity of the PP complex (1 nM) was analyzed at the indicated ATP concentrations in the absence or presence of CST (100 nM). Poly-dT was included at 300 nM, and P32-ATP was included at 12 µCi nmole−1 and 2.4 µCi nmole−1 for the 20 µM and 400 µM ATP reactions, respectively. Size standards in this and several other assays include both ssDNA (19, 37, 55 and 97 nt) and ssRNA (rA10). Results (averages S.D.) from three independent assays were quantified and plotted. (b) The two steps of the coupled “primase-Klenow” assay are illustrated on the left, and total32P incorporations plotted on the right. As controls, the priming step of the reactions was performed using no ribonucleotides or GTP only. Data (averages S.D.) are from three independent sets of assays.(c) The DNA polymerase activity of PP (1 nM) was analyzed in the absence or presence of CST (100 nM) using as substrates “pre-primed” poly-dT/rA10 (150 nM and 450 nM). (d) The DNA polymerase activity of PP (1 nM) was analyzed in the absence or presence of CST (150 nM) using as substrates the poly-dT/oligo-rA synthesized by PP in a priming reaction. (e) The primase-to-polymerase switch efficiency of PP (2 nM) was analyzed in the absence or presence of CST (100 and 200 nM) at the indicated ATP concentrations. P32-ATP was included at 24 µCi nmole−1 and 1.2 µCi nmole−1 for the 20 µM and 400 µM ATP reactions, respectively. (Because the Km of the primase for ribonucleotides is ~150 µM8, higher specific activity of P32-ATP is needed to generate robust signals in the 20 µM ATP reactions). (f) The fractions of extendable RNA primers (i.e., 7–10 nt long) that were not lengthened by the polymerase into RNA-DNA chimeras in assays such as those shown in D were quantified and plotted. Data (averages S.D.) are from three independent sets of assays. (g) The total numbers of RNA and RNA-DNA products in assays such as those shown in D were calculated by summing the normalized intensities (normalized against the number of labeled rA in each product) and plotted. (For the RNA-DNA oligomer, we assume an average RNA length of 9.) Data (averages S.D.) are from three independent sets of assays.
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Related In: Results  -  Collection

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Figure 2: CST promotes RNA priming and the primase-to-polymerase transition(a) The primase activity of the PP complex (1 nM) was analyzed at the indicated ATP concentrations in the absence or presence of CST (100 nM). Poly-dT was included at 300 nM, and P32-ATP was included at 12 µCi nmole−1 and 2.4 µCi nmole−1 for the 20 µM and 400 µM ATP reactions, respectively. Size standards in this and several other assays include both ssDNA (19, 37, 55 and 97 nt) and ssRNA (rA10). Results (averages S.D.) from three independent assays were quantified and plotted. (b) The two steps of the coupled “primase-Klenow” assay are illustrated on the left, and total32P incorporations plotted on the right. As controls, the priming step of the reactions was performed using no ribonucleotides or GTP only. Data (averages S.D.) are from three independent sets of assays.(c) The DNA polymerase activity of PP (1 nM) was analyzed in the absence or presence of CST (100 nM) using as substrates “pre-primed” poly-dT/rA10 (150 nM and 450 nM). (d) The DNA polymerase activity of PP (1 nM) was analyzed in the absence or presence of CST (150 nM) using as substrates the poly-dT/oligo-rA synthesized by PP in a priming reaction. (e) The primase-to-polymerase switch efficiency of PP (2 nM) was analyzed in the absence or presence of CST (100 and 200 nM) at the indicated ATP concentrations. P32-ATP was included at 24 µCi nmole−1 and 1.2 µCi nmole−1 for the 20 µM and 400 µM ATP reactions, respectively. (Because the Km of the primase for ribonucleotides is ~150 µM8, higher specific activity of P32-ATP is needed to generate robust signals in the 20 µM ATP reactions). (f) The fractions of extendable RNA primers (i.e., 7–10 nt long) that were not lengthened by the polymerase into RNA-DNA chimeras in assays such as those shown in D were quantified and plotted. Data (averages S.D.) are from three independent sets of assays. (g) The total numbers of RNA and RNA-DNA products in assays such as those shown in D were calculated by summing the normalized intensities (normalized against the number of labeled rA in each product) and plotted. (For the RNA-DNA oligomer, we assume an average RNA length of 9.) Data (averages S.D.) are from three independent sets of assays.
Mentions: A priori, the stimulatory effect of CST on total product synthesis can be due to an enhancement of the primase or DNA polymerase reaction, or the “coupling” between the two active sites. To distinguish between these possibilities, we first analyzed the effect of CST in separate primase and DNA polymerase reactions. For the primase-only assay, PP was incubated with poly-dT in the presence of labeled ATP as the sole nucleotide. Previous studies indicate that eukaryotic primase preferentially synthesizes unit-length primers (7–10 nt) or multiples thereof (i.e., multiples of 7–10 nt) in the absence of dNTP. Indeed, we observed both unit-length primers and dimers in our assays (Fig. 2a). More interestingly, the effect of CST in this assay was ATP concentration-dependent: CST stimulated primer synthesis by 2–3 fold in 20 µM ATP, but had much less effect in 400 µM ATP (Fig. 2a). As a complementary test, we performed a standard primase assay, which combines primer synthesis (using unlabeled 3 mM ATP) with extension by Klenow (using labeled dATP)2. Because Klenow was included in excess, the dATP incorporation was proportional to the RNA primers produced in the reaction. Using this assay, we found little stimulation of primase activity by CST (Fig. 2b). Thus, CST preferentially enhances primase activity at low ATP concentrations. We then performed polymerase-only assays, for which we used “pre-primed” Poly-dT/rA10 template and dATP as the substrate (Fig. 2c). No enhancement of DNA synthesis was observed in the presence of CST, indicating that the complex does not stimulate the DNA polymerase activity. We reasoned that the DNA template/RNA primer generated by primase might behave differently from the synthetic Poly-dT/rA10 substrate, and hence performed an alternative assay. In this assay, we first allowed PP to synthesize unlabeled oligo-rA using ATP as the sole nucleotide substrate. Upon completion of RNA synthesis, free ATP was removed from the reaction, and the resulting Poly-dT/oligo-rA mixture used as the substrate for DNA synthesis by PP in the absence or presence of CST (Fig. 2d). CST again had no effect on DNA synthesis, indicating that it does not stimulate “isolated” DNA polymerase α 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
Related in: MedlinePlus