The presented work provides a potential resolution to the apparent conundrum of how proteins with identical binding specificities coordinately function at a single site. Often DNA binding proteins form long-lived complexes with target DNAs in vitro that would interfere with the assembly of other structures and telomere-protein assemblies are no exception2
. For example, a stable Cdc13-telomere association at the extreme 3′ DNA end would block telomerase DNA binding yet Cdc13 binding at the extreme termini likely is required to protect the DNA from degradation in vivo. Our data supports a model in which Hsp82 frees the DNA end of competing binding proteins without interfering with critical regulatory events.
In our model, telomerase recruitment to a telomere initiates through transient contacts between Cdc13 and the holoenzyme (). To permit proper telomerase DNA association Hsp82 displaces proteins bound at the extreme 3′ DNA end including Cdc13. Recent genetic studies highlight the potential importance of maintaining a dynamic telomere environment, as the peak of telomere association by Cdc13, Stn1 and Est1 all occur in S-phase17,23
. Presumably, both capping (Stn1) and extending (Est1) proteins are telomere recruited each S-phase since not all telomeres are extended each cell cycle24
. Hence, depending upon the DNA length of a particular telomere a molecular choice is made to either permit or preclude telomerase function (i.e
., build an Est1-extending or Stn1-capping complex). If Hsp82 maintains Cdc13 in a dynamic DNA binding cycle, then any modifications (e.g.
, phosphorylation) meant to guide a telomere to a distinct operative phase would be immediately incorporated.
Figure 7 Hsp82 mediates telomere-protein dynamics. Cdc13 recruits select proteins to telomeric DNA including Stn1/Ten1 and telomerase/Est1. As the independent structures form stable and long-lived interactions with telomeric DNA, transitions between the distinct (more ...)
Recent genetic studies suggest post-translational modifications mediated by the Tel1 (ATM homolog) and Cdk1 kinases are critical for proper telomere DNA maintenance25,26
. For example, Tel1 is recruited to critically short telomeric DNA tracts, which are preferentially elongated, and Cdc13 has been shown to be a Tel1 target 25,27
. In addition, Cdk1 phosphorylates Cdc13 and the modification appears to influence the preference between Stn1- and Est1-containing Cdc13 nucleated telomere complexes26
. Hence, depending upon the Cdc13 phosphorylation-state, telomeric DNA would be in a Cdc13-Stn1/Ten1 unextendable state or a Cdc13-Est1/telomerase extendable form. Dynamic Cdc13 action, mediated by Hsp82, would enable the telomere system to rapidly transition between the different structures as needed.
In addition to protein dynamics, our studies reveal a Cdc13 telomerase activation function, which may account for the telomere lengthening phenotypes for several cdc13
. Of note, the Cdc13 telomerase stimulation activity that we observe is in stark contrast to a recent report showing telomerase inhibition by full-length Cdc1328
. Currently, the reason for the different effect of full-length Cdc13 on telomerase activity is unclear. We did find that the Cdc13 activation function was lost following a brief (5 min) incubation at 42°C while DNA binding activity was unaffected (data not shown), which suggests that the conformation of the activation surface is labile. Perhaps by expressing full-length Cdc13 in a specialized bacterial strain (i.e
., streptomycin-resistant strain), which was critical for obtaining soluble recombinant protein29
, the activation surface was provided an opportunity to properly fold. Opposed to the data on full-length Cdc13, both studies did observe telomerase inhibition by the Cdc13 DNA binding domain fragment and the DBD displayed no apparent difference when expressed in either classic or streptomycin-resistant bacterial cells29
Nevertheless, prior genetic data suggests a role for Cdc13 with telomerase that is downstream of telomere nucleation. For example, the cdc13-4
yeast have short but stable telomeric DNA despite an apparent capacity of Cdc13-4 to recruit telomerase to telomeres and protect the DNA from degradation30
. While the effected residue (P235S) occurs within the amino-terminal domain, the mechanism for the telomere defect was not identified. However, the in vivo phenotype is consistent with a post-recruitment function for Cdc1330
. Intriguingly, the Cdc13-109 and Cdc13-231 mutants lead to over-elongated telomeric DNA despite a decline in DNA binding activity5
. If the sole positive function of Cdc13 was to recruit telomerase to a telomere, then a decline in DNA binding should result in shorter not longer telomeric DNA. Additionally, the hyper-elongation of telomeric DNA in the cdc13-5
yeast is consistent with a post-recruitment role for Cdc13. The Cdc13-5 protein (N-domain) consists of the amino-terminus and DNA binding domain6
. In the absence of the Cdc13 carboxyl-terminal Stn1 interaction site the telomeric DNA should be vulnerable to nuclease attack. Yet, the telomeric DNA is not degraded in the cdc13-5
yeast but rather is over-elongated6
. We suggest that, in addition to recruiting telomerase, Cdc13-5 activates telomerase to not only hyper-extend the DNA but also to counter any potential DNA degradation. Of note, the stimulatory function is downstream from Cdc13-mediated recruitment since the effect is abrogated when combined with the Cdc13 point mutation cdc13-2
. Taken together, established genetic data support our contention that Cdc13 modulates the DNA-bound enzyme. We suggest that telomerase cofactors, including Cdc13, likely reengage the telomere protein assembly by tethering to the DNA-bound telomerase enzyme.
A chaperone-mediated protein dynamics model has been previously proposed for transcription pathways31
. In brief, molecular chaperones promote a dynamic action for transcription factors that is necessary to permit rapid functional recruitment of multiple coactivating complexes to a gene promoter. An important distinction, however, is the impact of the Hsp90 chaperone: Hsp90 promotes DNA binding by transcription factors but facilitates dissociation of Cdc13-DNA complexes. Perhaps the dual role for Hsp82 at telomeres provides an explanation for the difference. By exploiting the telomerase-associated cofactor Hsp82 to both support telomerase function and to clear the telomeric DNA of competing proteins, an elegant means to ensure telomeric DNA extension within the short working period allotted telomerase at the end of S phase is provided.
Traditionally Hsp90 has been viewed as a cytoplasmic molecular chaperone required for the late folding stages of signaling molecules32
. However, recent studies including high-throughput screens have identified a broad-range of potential nuclear client proteins2
. Given the impact of Hsp90/Hsp82 both on telomeric and transcriptional targets, there appears to be a general cellular role for Hsp90 chaperones in controlling protein-DNA dynamics. In brief, multi-step pathways, including the telomere system, move forward through high affinity interactions between the low abundant proteins unique to that system (e.g
., Cdc13, telomerase, Stn1) while proper structure composition (i.e
., competitive interactions) along with efficient transitions between the different assemblies are mediated by transient, low affinity interactions with the highly abundant molecular chaperones.