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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

The physical interaction between Stn1 and Pol12(a) A schematic depiction of the fusion tags used for the co-expression/pull down assays is shown at the top. Fractions derived from Ni-NTA purification of extracts containing Stn1 alone or Stn1 and Pol12 were subjected to anti-FLAG (M2) affinity purification. The input and purified fractions were subjected to SDS-PAGE, Coomassie staining, and Western analysis using the indicated antibodies. Two proteolytic fragments of Pol12 detected by anti-FLAG Western are indicated by asterisks. (b) Extracts from strains expressing Stn1 domains alone or in combination with Pol12 were subjected to M2 affinity purification, and the input and purified fractions analyzed by SDS-PAGE and Coomassie staining. (c) Extracts from strains expressing Stn1C WH1 or WH2 motifs alone or in combination with Pol12 were subjected to GST affinity purification, and the input and purified fractions analyzed by Western. Pol12 was detected by anti-FLAG, whereas WH1 and WH2 anti-HIS antibodies.
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Figure 4: The physical interaction between Stn1 and Pol12(a) A schematic depiction of the fusion tags used for the co-expression/pull down assays is shown at the top. Fractions derived from Ni-NTA purification of extracts containing Stn1 alone or Stn1 and Pol12 were subjected to anti-FLAG (M2) affinity purification. The input and purified fractions were subjected to SDS-PAGE, Coomassie staining, and Western analysis using the indicated antibodies. Two proteolytic fragments of Pol12 detected by anti-FLAG Western are indicated by asterisks. (b) Extracts from strains expressing Stn1 domains alone or in combination with Pol12 were subjected to M2 affinity purification, and the input and purified fractions analyzed by SDS-PAGE and Coomassie staining. (c) Extracts from strains expressing Stn1C WH1 or WH2 motifs alone or in combination with Pol12 were subjected to GST affinity purification, and the input and purified fractions analyzed by Western. Pol12 was detected by anti-FLAG, whereas WH1 and WH2 anti-HIS antibodies.

Mentions: Previous analysis points to two possible contacts between CST and PP: one between Cdc13 and Pol1 and the other between Stn1 and Pol1219,45,46. That Stn1 alone but not Cdc13 is able to stimulate PP suggests that the latter interaction may be functionally more important. We therefore examined the physical interaction between Stn1 and Pol12 using a co-expression/affinity purification assay. The two proteins were expressed separately or together in E. coli as His6-SUMO and GST-FLAG fusion proteins, respectively. Extracts were prepared and subjected to affinity purification to assess complex formation. As hypothesized, His6-SUMO-Stn1 and GST-FLAG-Pol12 remained stably associated with each other following both Ni-NTA and anti-FLAG affinity chromatography (Fig. 4a). Moreover, the two polypeptides are present at a 1:1 ratio, suggesting a 1:1 stoichiometry. Surprisingly, when Stn1 and Pol12 were first separately purified, and then subjected to anti-FLAG or Glutathione pull down assays, little interaction was detected between the two proteins. This observation suggested that one of the proteins may adopt an unproductive conformation when it is expressed in the absence of the other. Because Stn1 alone is active in PP stimulation, we suspect that isolated Pol12 may adopt an aberrant conformation. Indeed, an existing crystal structure of the ScPol1-Pol12 complex revealed extensive contacts between Pol12 and the C-terminus of Pol147, suggesting that Pol1 may influence Pol12 conformation. We then tested the N-and C-terminal domains of Stn1 and found that each domain can independently bind Pol12 (Fig. 4b). Thus, the ability of both the N- and C-terminus of Stn1 to stimulate PP activity correlates with their binding to Pol12.


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)

The physical interaction between Stn1 and Pol12(a) A schematic depiction of the fusion tags used for the co-expression/pull down assays is shown at the top. Fractions derived from Ni-NTA purification of extracts containing Stn1 alone or Stn1 and Pol12 were subjected to anti-FLAG (M2) affinity purification. The input and purified fractions were subjected to SDS-PAGE, Coomassie staining, and Western analysis using the indicated antibodies. Two proteolytic fragments of Pol12 detected by anti-FLAG Western are indicated by asterisks. (b) Extracts from strains expressing Stn1 domains alone or in combination with Pol12 were subjected to M2 affinity purification, and the input and purified fractions analyzed by SDS-PAGE and Coomassie staining. (c) Extracts from strains expressing Stn1C WH1 or WH2 motifs alone or in combination with Pol12 were subjected to GST affinity purification, and the input and purified fractions analyzed by Western. Pol12 was detected by anti-FLAG, whereas WH1 and WH2 anti-HIS antibodies.
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Related In: Results  -  Collection

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Figure 4: The physical interaction between Stn1 and Pol12(a) A schematic depiction of the fusion tags used for the co-expression/pull down assays is shown at the top. Fractions derived from Ni-NTA purification of extracts containing Stn1 alone or Stn1 and Pol12 were subjected to anti-FLAG (M2) affinity purification. The input and purified fractions were subjected to SDS-PAGE, Coomassie staining, and Western analysis using the indicated antibodies. Two proteolytic fragments of Pol12 detected by anti-FLAG Western are indicated by asterisks. (b) Extracts from strains expressing Stn1 domains alone or in combination with Pol12 were subjected to M2 affinity purification, and the input and purified fractions analyzed by SDS-PAGE and Coomassie staining. (c) Extracts from strains expressing Stn1C WH1 or WH2 motifs alone or in combination with Pol12 were subjected to GST affinity purification, and the input and purified fractions analyzed by Western. Pol12 was detected by anti-FLAG, whereas WH1 and WH2 anti-HIS antibodies.
Mentions: Previous analysis points to two possible contacts between CST and PP: one between Cdc13 and Pol1 and the other between Stn1 and Pol1219,45,46. That Stn1 alone but not Cdc13 is able to stimulate PP suggests that the latter interaction may be functionally more important. We therefore examined the physical interaction between Stn1 and Pol12 using a co-expression/affinity purification assay. The two proteins were expressed separately or together in E. coli as His6-SUMO and GST-FLAG fusion proteins, respectively. Extracts were prepared and subjected to affinity purification to assess complex formation. As hypothesized, His6-SUMO-Stn1 and GST-FLAG-Pol12 remained stably associated with each other following both Ni-NTA and anti-FLAG affinity chromatography (Fig. 4a). Moreover, the two polypeptides are present at a 1:1 ratio, suggesting a 1:1 stoichiometry. Surprisingly, when Stn1 and Pol12 were first separately purified, and then subjected to anti-FLAG or Glutathione pull down assays, little interaction was detected between the two proteins. This observation suggested that one of the proteins may adopt an unproductive conformation when it is expressed in the absence of the other. Because Stn1 alone is active in PP stimulation, we suspect that isolated Pol12 may adopt an aberrant conformation. Indeed, an existing crystal structure of the ScPol1-Pol12 complex revealed extensive contacts between Pol12 and the C-terminus of Pol147, suggesting that Pol1 may influence Pol12 conformation. We then tested the N-and C-terminal domains of Stn1 and found that each domain can independently bind Pol12 (Fig. 4b). Thus, the ability of both the N- and C-terminus of Stn1 to stimulate PP activity correlates with their binding to Pol12.

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