Limits...
POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis.

Renfrew KB, Song X, Lee JR, Arora A, Shippen DE - PLoS Genet. (2014)

Bottom Line: We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo.Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity.Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Protection of Telomeres 1 (POT1) is a conserved nucleic acid binding protein implicated in both telomere replication and chromosome end protection. We previously showed that Arabidopsis thaliana POT1a associates with the TER1 telomerase RNP, and is required for telomere length maintenance in vivo. Here we further dissect the function of POT1a and explore its interplay with the CST (CTC1/STN1/TEN1) telomere complex. Analysis of pot1a mutants revealed that POT1a is not required for telomerase recruitment to telomeres, but is required for telomerase to maintain telomere tracts. We show that POT1a stimulates the synthesis of long telomere repeat arrays by telomerase, likely by enhancing repeat addition processivity. We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo. Unexpectedly, the in vitro interaction of STN1 with TEN1 and POT1a was mutually exclusive, indicating that POT1a and TEN1 may compete for the same binding site on STN1 in vivo. Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity. Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

No MeSH data available.


A model for telomere replication in Arabidopsis.In the un-extendable state, telomeres are bound by the heterotrimeric CST complex. The telomerase RNP is positioned at the chromosome terminus by an unknown recruitment factor (X) during S phase. TEN1 is displaced. POT1a (Pa) contacts STN1 (S) and CTC1 (C) to promote a telomere extendable state. POT1a also stimulates telomerase enzymatic properties. TEN1 represses telomerase activity and thus may help to terminate telomerase action. Telomerase is removed and replaced by POLα for C-strand fill-in and terminal DNA processing. The telomere is then converted into an un-extendable state.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4199523&req=5

pgen-1004738-g007: A model for telomere replication in Arabidopsis.In the un-extendable state, telomeres are bound by the heterotrimeric CST complex. The telomerase RNP is positioned at the chromosome terminus by an unknown recruitment factor (X) during S phase. TEN1 is displaced. POT1a (Pa) contacts STN1 (S) and CTC1 (C) to promote a telomere extendable state. POT1a also stimulates telomerase enzymatic properties. TEN1 represses telomerase activity and thus may help to terminate telomerase action. Telomerase is removed and replaced by POLα for C-strand fill-in and terminal DNA processing. The telomere is then converted into an un-extendable state.

Mentions: Altogether, our data suggest a model in which POT1a facilitates telomere maintenance in two ways: by promoting the switch from the un-extendable to the extendable state and by stimulating telomerase enzyme activity (Fig. 7). In S phase, telomerase holoenzyme is recruited to the G-overhang through an unknown mechanism. The enzyme associates with CTC1 and STN1 through contacts with POT1a, and POT1a stimulates G-strand synthesis. One attractive hypothesis is that mobilization of POT1a to the chromosome terminus triggers the exchange of the telomerase negative regulator TEN1 from STN1 as part of the switch to the telomerase extendable state. Although our in vitro data indicate that STN1 has a higher affinity for TEN1 than POT1a OB1, additional contacts by other regions of POT1a or between POT1a and CTC1 may stabilize its interaction with STN1. Furthermore, shifting telomerase-CST interactions are likely to be governed by cell cycle specific posttranslational modifications such as those described for yeast Est1 and CST, as well as human TPP1 [19], [20], [37]. Once the G-strand is extended, telomerase action is terminated, perhaps with the assistance of TEN1. This clears the way for conventional replication machinery and processing enzymes to complete telomere replication and return the telomere to its fully protected un-extendable state. Although additional studies are needed to precisely delineate the telomere-telomerase interface and its control during telomere replication, our findings underscore the highly dynamic nature of telomerase-telomere transactions and suggest that modulation of telomerase enzyme activity at the chromosome terminus contributes to the bimodal switch in telomere states.


POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis.

Renfrew KB, Song X, Lee JR, Arora A, Shippen DE - PLoS Genet. (2014)

A model for telomere replication in Arabidopsis.In the un-extendable state, telomeres are bound by the heterotrimeric CST complex. The telomerase RNP is positioned at the chromosome terminus by an unknown recruitment factor (X) during S phase. TEN1 is displaced. POT1a (Pa) contacts STN1 (S) and CTC1 (C) to promote a telomere extendable state. POT1a also stimulates telomerase enzymatic properties. TEN1 represses telomerase activity and thus may help to terminate telomerase action. Telomerase is removed and replaced by POLα for C-strand fill-in and terminal DNA processing. The telomere is then converted into an un-extendable state.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199523&req=5

pgen-1004738-g007: A model for telomere replication in Arabidopsis.In the un-extendable state, telomeres are bound by the heterotrimeric CST complex. The telomerase RNP is positioned at the chromosome terminus by an unknown recruitment factor (X) during S phase. TEN1 is displaced. POT1a (Pa) contacts STN1 (S) and CTC1 (C) to promote a telomere extendable state. POT1a also stimulates telomerase enzymatic properties. TEN1 represses telomerase activity and thus may help to terminate telomerase action. Telomerase is removed and replaced by POLα for C-strand fill-in and terminal DNA processing. The telomere is then converted into an un-extendable state.
Mentions: Altogether, our data suggest a model in which POT1a facilitates telomere maintenance in two ways: by promoting the switch from the un-extendable to the extendable state and by stimulating telomerase enzyme activity (Fig. 7). In S phase, telomerase holoenzyme is recruited to the G-overhang through an unknown mechanism. The enzyme associates with CTC1 and STN1 through contacts with POT1a, and POT1a stimulates G-strand synthesis. One attractive hypothesis is that mobilization of POT1a to the chromosome terminus triggers the exchange of the telomerase negative regulator TEN1 from STN1 as part of the switch to the telomerase extendable state. Although our in vitro data indicate that STN1 has a higher affinity for TEN1 than POT1a OB1, additional contacts by other regions of POT1a or between POT1a and CTC1 may stabilize its interaction with STN1. Furthermore, shifting telomerase-CST interactions are likely to be governed by cell cycle specific posttranslational modifications such as those described for yeast Est1 and CST, as well as human TPP1 [19], [20], [37]. Once the G-strand is extended, telomerase action is terminated, perhaps with the assistance of TEN1. This clears the way for conventional replication machinery and processing enzymes to complete telomere replication and return the telomere to its fully protected un-extendable state. Although additional studies are needed to precisely delineate the telomere-telomerase interface and its control during telomere replication, our findings underscore the highly dynamic nature of telomerase-telomere transactions and suggest that modulation of telomerase enzyme activity at the chromosome terminus contributes to the bimodal switch in telomere states.

Bottom Line: We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo.Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity.Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT
Protection of Telomeres 1 (POT1) is a conserved nucleic acid binding protein implicated in both telomere replication and chromosome end protection. We previously showed that Arabidopsis thaliana POT1a associates with the TER1 telomerase RNP, and is required for telomere length maintenance in vivo. Here we further dissect the function of POT1a and explore its interplay with the CST (CTC1/STN1/TEN1) telomere complex. Analysis of pot1a mutants revealed that POT1a is not required for telomerase recruitment to telomeres, but is required for telomerase to maintain telomere tracts. We show that POT1a stimulates the synthesis of long telomere repeat arrays by telomerase, likely by enhancing repeat addition processivity. We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo. Unexpectedly, the in vitro interaction of STN1 with TEN1 and POT1a was mutually exclusive, indicating that POT1a and TEN1 may compete for the same binding site on STN1 in vivo. Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity. Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

No MeSH data available.