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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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The intra-species and inter-species binding and stimulatory activities of C. glabrata and human Stn1(a) HsPP and CgPP were analyzed in the absence or presence of HsSTN1 or CgStn1 using the poly-dT template, unlabeled ATP and labeled dATP. (b) HsPP and CgPP were analyzed in the absence or presence of HsSTN1 or CgStn1 using the poly-dT template, labeled ATP and unlabeled dATP.(c) Extracts from strains expressing the indicated combinations of Pol12 and Stn1 were subjected to GST affinity purification, and the extracts and purified fractions analyzed by Western using anti-HIS and anti-GST antibodies.
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Figure 5: The intra-species and inter-species binding and stimulatory activities of C. glabrata and human Stn1(a) HsPP and CgPP were analyzed in the absence or presence of HsSTN1 or CgStn1 using the poly-dT template, unlabeled ATP and labeled dATP. (b) HsPP and CgPP were analyzed in the absence or presence of HsSTN1 or CgStn1 using the poly-dT template, labeled ATP and unlabeled dATP.(c) Extracts from strains expressing the indicated combinations of Pol12 and Stn1 were subjected to GST affinity purification, and the extracts and purified fractions analyzed by Western using anti-HIS and anti-GST antibodies.

Mentions: Even though mammalian CST is known to bind and stimulate mammalian PP, the mechanistic basis of this regulation is largely undefined. To determine if the mechanisms we defined in fungi are applicable to other organisms, we examined the activities of purified human PP (HsPP) and human STN1 (HsSTN1) using the same assays. In the standard coupled assays on the poly-dT template, HsPP generated chimeric products that are slightly longer than those of CgPP, but otherwise displayed similar properties with respect to template and nucleotide utilization (Fig. 5a, lane 1 and 4). In general agreement with the fungal studies, hsSTN1 alone stimulated product synthesis by hsPP and increased the lengths of the RNA-DNA chimera slightly (Fig. 5a, lane 1 and 2). Likewise, in the primase-to-polymerase switch assays, hsSTN1 increased the total number of RNA and RNA-DNA oligomers (by ~70%), and reduced the fraction of the RNA primers that terminated at the priming stage (by ~30%) (Fig. 5b). (Note that while the levels of un-extended RNA oligomers were similar in lane 1, 2 and 3, the higher levels of total reaction products in lane 2 and 3 indicate that the fractions of un-extended RNA primers were lower for these two reactions.) Remarkably, despite the very low level of sequence similarity between CgStn1 and Human STN1 (HsSTN1) (~17% identity and 33% similarity), both proteins displayed substantial stimulatory activities on the heterologous PP complex (Fig. 5a). In a side-by-side comparison of the two proteins in Poly-dT assays over a range of concentrations, HsSTN1 is only about 2-fold less active than CgStn1 in stimulating CgPP (Supplementary Fig. 6d).


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 intra-species and inter-species binding and stimulatory activities of C. glabrata and human Stn1(a) HsPP and CgPP were analyzed in the absence or presence of HsSTN1 or CgStn1 using the poly-dT template, unlabeled ATP and labeled dATP. (b) HsPP and CgPP were analyzed in the absence or presence of HsSTN1 or CgStn1 using the poly-dT template, labeled ATP and unlabeled dATP.(c) Extracts from strains expressing the indicated combinations of Pol12 and Stn1 were subjected to GST affinity purification, and the extracts and purified fractions analyzed by Western using anti-HIS and anti-GST antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4269169&req=5

Figure 5: The intra-species and inter-species binding and stimulatory activities of C. glabrata and human Stn1(a) HsPP and CgPP were analyzed in the absence or presence of HsSTN1 or CgStn1 using the poly-dT template, unlabeled ATP and labeled dATP. (b) HsPP and CgPP were analyzed in the absence or presence of HsSTN1 or CgStn1 using the poly-dT template, labeled ATP and unlabeled dATP.(c) Extracts from strains expressing the indicated combinations of Pol12 and Stn1 were subjected to GST affinity purification, and the extracts and purified fractions analyzed by Western using anti-HIS and anti-GST antibodies.
Mentions: Even though mammalian CST is known to bind and stimulate mammalian PP, the mechanistic basis of this regulation is largely undefined. To determine if the mechanisms we defined in fungi are applicable to other organisms, we examined the activities of purified human PP (HsPP) and human STN1 (HsSTN1) using the same assays. In the standard coupled assays on the poly-dT template, HsPP generated chimeric products that are slightly longer than those of CgPP, but otherwise displayed similar properties with respect to template and nucleotide utilization (Fig. 5a, lane 1 and 4). In general agreement with the fungal studies, hsSTN1 alone stimulated product synthesis by hsPP and increased the lengths of the RNA-DNA chimera slightly (Fig. 5a, lane 1 and 2). Likewise, in the primase-to-polymerase switch assays, hsSTN1 increased the total number of RNA and RNA-DNA oligomers (by ~70%), and reduced the fraction of the RNA primers that terminated at the priming stage (by ~30%) (Fig. 5b). (Note that while the levels of un-extended RNA oligomers were similar in lane 1, 2 and 3, the higher levels of total reaction products in lane 2 and 3 indicate that the fractions of un-extended RNA primers were lower for these two reactions.) Remarkably, despite the very low level of sequence similarity between CgStn1 and Human STN1 (HsSTN1) (~17% identity and 33% similarity), both proteins displayed substantial stimulatory activities on the heterologous PP complex (Fig. 5a). In a side-by-side comparison of the two proteins in Poly-dT assays over a range of concentrations, HsSTN1 is only about 2-fold less active than CgStn1 in stimulating CgPP (Supplementary Fig. 6d).

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