To understand how human POT1 recognized and bound to the telomere, the researchers crystallized a form of POT1 bound to the critical ten-nucleotide segment of DNA. They then used x-ray diffraction to reveal the structure of the complex. Unexpectedly, they found that unlike the yeast version of the protein, human POT1 contained two distinct OB-folds. The grooves of the two folds align with one another, forming a continuous channel where the telomeric DNA can fit.
They also learned that while the protein would bind to a ten-nucleotide sequence, the structure could also accommodate twelve nucleotides. "So it turns out it doesn't bind one six, it binds two times six," Cech said. On a single chromosome end, he said, there might be eight to 24 POT1 molecules coating the DNA tail.
The structure of the complex suggests that the end of the chromosome is tightly protected by POT1, and the researchers were able to verify this with additional biochemical experiments. When the POT1-DNA complex was treated with a solution that would normally modify the DNA at specific sites, no such changes occurred indicating that those sites were completely enclosed by the POT1 protein.
According to Cech, the findings raise important questions about the regulation of telomerase. When telomeric DNA is buried within POT1, telomerase cannot access the DNA to elongate the telomere. "This is something that could keep the cell from making telomeres all day long," he said. "We think this is one level at which telomerase is regulated." Therefore, he said, an important next step will be to determine the cellular mechanism that switches the telomere to the on state so that elongation can occur.
Contact: Jennifer Michalowski
Howard Hughes Medical Institute