Telomeres play an essential role in maintaining the integrity of the chromosome, protecting it from degradation and from end-to-end fusion with other chromosomes. They also serve as substrates for the enzyme telomerase, which is reponsible for replicating the end region in cell division. The DNA component of telomeres usually begins with a double-stranded region consisting of repeated sequences of five to nine base pairs, where the number of base pairs varies from species to species. It then ends with a single-stranded region rich in guanine (G) and thymine (T) bases called a 3´ overhang that bends back on itself to facilitate a protective t-loop structure.

Structure of the Pot1pN–ssDNA complex. Ribbon diagram of Pot1pN exhibits β-strands (cyan), α-helices (green), and loops (red). The DNA, shown in a stick representation with carbon (white), nitrogen (blue), oxygen (red), and phosphorus (yellow), fits into a groove formed by one side of the β barrel and two protruding loops.

Protecting Chromosome Ends

Chromosomes in the nuclei of cells contain DNA with the recipe for life, so it is fortunate indeed that nature has devised ways to protect chromosomes from damage and repair them when things do go awry. Chromosomes in plants and animals are linear, raising questions such as how does the cell tell the difference between the end of a normal chromosome and a break to be repaired in a damaged one, and what keeps the ends of neighboring chromosomes from sticking together? Part of the answer comes in the form of telomeres, highly specialized complexes of DNA and protein molecules that form end caps for chromosomes.

Among the features of telomeres are regions at the very tip of single- rather than double-stranded DNA. Since the normal DNA-replication process during cell division doesn't work in this region, an enzyme called telomerase takes care of this. Protection of telomeres (Pot1) proteins have now been found in organisms ranging from yeasts to humans and appear to be essential to the end-capping process. They have also been proposed to be part of the process by which the telomere is made available to telomerase during replication. Lei et al. have obtained a high-resolution structure for the portion of a Pot1 protein with a region of single-stranded DNA in yeast. The details of their structure provide a framework for understanding telomere functions at the molecular level.

Among proteins specific for this single-stranded overhang, Pot1 proteins provide the most widespread solution to chromosome end-capping in eukaryotes. Previous biochemical work by the group showed that the N-terminal DNA-binding domain Pot1pN in the fission yeast Schizosaccharomyces pombe, a popular model organism, had a sequence similarity to the first OB (oligonucleotide/oligosaccharide binding) fold of the α subunit of the telomere end-binding protein of the ciliated protozoan Oxytricha nova. However, without any structural information, the molecular basis for the DNA-binding specificity that ensures that Pot1 only binds to single-stranded G-rich telomeric DNA (ssDNA) was not revealed.

To explore the molecular mechanism of this binding specificity, the researchers crystallized Pot1pN in three different forms in complex with either the pentanucleotide GGTTA or the hexanucleotide GGTTAC. From data obtained at ALS Beamlines 8.2.1 and 8.2.2, they solved the structure of one Pot1pN–GGTTA crystal form by means of single-wavelength anomalous dispersion (SAD) and refined the structure to a resolution of 1.9 Å. Then they determined the structures of the other two crystal forms by molecular replacement (MR) and a searching model without the ssDNA. They found that the structures were essentially identical in all three crystal forms.

SsDNA self-recognition by G-T base pairing interactions and binding with the protein. Owing to the ssDNA intramolecular hydrogen bonds (dotted yellow lines), the donor/acceptor groups of the bases face the inner side of the binding groove, thus encouraging extensive intermolecular hydrogen bonding interactions with the protein (dotted green lines).

The crystal structure shows that Pot1pN consists of a compact single domain, the OB fold, comprising a highly curved, five-stranded, antiparallel β barrel, as implied by the sequence alignment. The single-stranded DNA binds in a basic concave groove, a characteristic of OB-fold proteins, formed by one side of the β barrel and two protruding loops.

More important, the structure explains the exceptionally high sequence specificity of protein binding. An unanticipated ssDNA-self recognition involving novel G-T base pairing compacts the DNA, and this folded DNA structure is bound by the protein through stacking and hydrogen bonding. Any base sequence change would disrupt the ability of the DNA to fold into this structure, thus preventing it from contacting the array of protein hydrogen-bonding groups. Mutational analysis established the in vivo biological importance of the Pot1–ssDNA interaction by showing that two residues that were already implicated by the structure in DNA binding are also important for telomere maintenance and cell survival.

The in vivo biological importance of the Pot1–ssDNA interaction. Mutational analysis of residues important for the interaction of Pot1pN (blue) and ssDNA (yellow) showed that the red mutations, T62V and F88A, already implicated by the structure in DNA binding, are also important for telomere maintenance and cell survival. The mutated residues are shown as green and red ball-and-stick models.

The structure will serve as a framework for understanding telomere functions at the molecular level. These functions include Pot1 protein's essential role in chromosome end-capping in S. pombe, its contribution to regulation of telomerase in human cells, and its proposed role in switching telomeres between the protective t-loop and an open structure accessible to telomerase during replication.

Research conducted by M. Lei, E.R. Podell, and T.R. Cech (University of Colorado) and P. Baumann (Stowers Institute for Medical Research).

Research funding: The National Institutes of Health, the Stowers Institute for Medical Research, and the Helen Hay Whitney Foundation. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: M. Lei, E.R. Podell, P. Baumann, and T.R. Cech, "DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA," Nature 426, 198 (2003).

ALSNews Vol. 240, April 28, 2004