Telomere and Telomerase

DNA is the carrier of genetic information. In the cell, each DNA molecule is packaged in a structure called chromosome. The ends of linear chromosomes are capped by structures known as telomeres to prevent fusion with neighboring chromosomes. For example, the simplest type of telomere is a covalently closed DNA hairpin structure. Telomeres are maintained by an enzyme called telomerase during DNA replication.

Replication of a linear chromosome with hairpin telomeres (Molecular Cell, Volume 27, Pages 901-913)

Protelomerase TelK

TelK localizes two 56-bp target sequences at opposite poles of the DNA circle replication intermediate, and it generates hairpin-capped ends via site-specific excision, strand exchange and re-ligation. TelK-induced DNA hairpin formation occurs independently of adenosine triphosphate (ATP) or other cofactors such as Mg2+, and TelK is a single-turnover protein. TelK shares sequence and structural homology to tyrosine recombinases (Y-recombinases) and type IB topoisomerases. The crystal structure of TelK538, a truncation mutant of wild-type TelK, complexed with a 44mer target-DNA substrate shows two tightly bound TelK monomers that form the active dimer oriented head-to-head on the dyad symmetric DNA target site. Dimerization induces a sharp bend in the DNA substrate (see figure below).

Structure of the TelK Dimer Bound to DNA (Molecular Cell, Volume 27, Pages 901-913)

Question: How does TelK dimer quickly and reliably localize specific target sites (telomere sequences) on DNA

Although TelK is known to be a monomer in solution, target-sequence activity requires TelK to dimerize at its target site in the correct head-to-head orientation. The process by which site-specific proteins find their DNA target sites has remained poorly characterized for proteins that are functional on DNA as dimers or higher-order protein complexes but are monomeric in solution. To understand the target-site search mechanism for this protein, we studied the interaction of TelK with both DNA lacking and containing the target sequence.


TelK exhibits two distinct modes of interaction to non-target DNA

We first investigated the interaction between TelK and non-target DNA. We used TIRF microscopy to image individual QD-labelled proteins on linearly extended DNA that lacked the TelK target sequence. Before a typical measurement, QD-labelled TelK was flowed into the chamber and left to incubate with the DNA bridges for 5 min to allow TelK to bind to the DNA bridges. The positions of individual protein units on DNA were tracked by Gaussian-fitting the fluorescent intensity. Analysis over many DNA bridges revealed a clear bimodal distribution in the population of fluorescently labelled protein, showing species of mobile and immobile spots. TelK monomers diffuse along non-target DNA, whereas dimers immobilize.

Two DNA binding modes of TelK.

Dimer-induced condensation of non-target DNA by TelK causes protein immobilization

difference TelK dimers condense non-target DNA.

What is the mechanism for the observed dimer immobilization? Crystal structures of the TelK dimer in complex with target DNA indicate a conformation in which monomer-monomer contacts and a tight interaction to the target sequence kink the DNA by an angle of 73 degree. Dimer immobilization observed in our TIRF measurements suggests that similar monomer-monomer contacts may also be responsible for dimer immobilization on non-target DNA. To test this hypothesis, we next used an optical trap to probe the TelK-DNA conformation on an extended DNA molecule. Despite the absence of the TelK target site on the DNA, we repeatedly observed the DNA end-to-end extension to decrease by a few nanometres on exposure to TelK. In contrast, control experiments performed in buffer lacking TelK showed no DNA condensation. Condensation behaviour is expected as a result of TelK dimer formation at the DNA target site, as the 73 degree bend of the target DNA observed in crystal structures condenses the DNA, reducing its end-to-end extension. However, DNA condensation in the absence of the target site is not expected. The observed condensation in the optical trap measurements suggests that the same dimer-DNA con- formation may be adopted on non-target DNA.

To test this suggestion, we performed a series of MD simulations to study the interaction of (i) a TelK dimer with target DNA, (ii) a TelK dimer with non-target DNA and (iii) a TelK monomer with non-target DNA. The simulations reveal that dimers induce near identical bend angles (70) onto DNA with and without the target sequence. We used the bend angle observed in these MD simulations to estimate the change in DNA end-to-end extension as a result of TelK dimerization on the non-target DNA substrate used in our optical trap assays. We compared with the extension of the condensed DNA to that of linearly extended DNA taking into account the bend angle observed in the MD simulation, the DNA elastic properties and the force at which the tether was held in the optical trap. Based on this calculation, we expect a condensation step size of 7.5 nm at a tension of 5.2 pN. As shown in figure, the observed step-size distribution is bimodal, with one peak at 7.2 nm, in excellent agreement with the prediction. This result indicates that a significant fraction of condensation events observed correspond to formation of DNA-TelK dimer complexes similar to those seen in crystal structures. Simulations of a TelK monomer binding to non-target DNA further show that the monomeric form of TelK is unable to bend DNA as a dimer does. Instead, monomeric TelK interaction with DNA results in a large decrease in the bend angle. A histogram of the end-to-end distance of the 44-bp DNA substrate shows significant condensation in the case of the TelK dimer, but on average no condensation for a TelK monomer on non-target DNA. MD results provide strong support for the claim that the condensation events observed in the optical trap measurements correspond to TelK dimerization.

MD simulations confirm that Telk dimers bend non-target DNA, whereas monomers cannot.

Monomeric TelK bind preferentially to the target DNA sequence

To determine whether dimers or monomers confer targetsite specificity, we considered a subset of our data by comparing only DNA tethers that showed one single blinking fluorescent spot per molecule of extended target or non-target DNA. For all singly occupied DNA bridges containing the target sequence, all observed blinking fluorescent spots were located at the target site, indicating that monomeric TelK has a binding preference for the target site.

TelK binds preferentially to target DNA.



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