Our long-term goal is to understand how the vertebrate telomeric complex executes its two main functions: to protect chromosome ends and to mediate their replication. Telomeres shield chromosome ends from extensive degradation and fusion and allow cells to discriminate between broken DNA and natural chromosome ends, thus ensuring that DNA damage checkpoints are not activated in undamaged cells.
A second challenge is to understand how telomeres are maintained. Due to the mechanism of DNA replication, small amounts of telomeric DNA are lost with each cell division. To make up for this terminal sequence loss, telomeres need to engage alternative replication strategies. In mammals, this problem is primarily solved by telomerase. Telomerase regulation and the dynamics of human telomeres have been implicated in cellular senescence and cancer.
We have isolated four human proteins and are studying the role of these factors in the protection and maintenance of chromosome ends. TRF1 and TRF2 are two related DNA-binding factors that coat along the length of the duplex telomeric repeat array and control telomere length. TRF1 interacts with a third telomeric protein, the telomeric poly(ADP-ribose)polymerase tankyrase, that acts as a negative regulator of TRF1. TRF2 binds to hRap1, the human ortholog of the yeast telomeric protein, Rap1p. Human Rap1, like its yeast counterpart, contributes to the control of telomere maintenance. However, certain differences between human and yeast Rap1 suggest a dramatic change in the telomeric complex during the evolution of the budding yeasts.
TRF2 plays an essential role in telomere protection. Inhibition of this factor leads to immediate deprotection of chromosome ends as evidenced by the loss of the telomeric 3´ tail, rapid fusion of chromosome ends and the activation of a DNA-damage response pathway. Thus, cells respond to telomeres devoid of TRF2 as if they resemble damaged DNA. An important goal is to understand the mechanism by which TRF2 protects chromosome ends.
One possible mechanism for telomere protection and the regulation of telomere maintenance is suggested by the observation that telomeres form large duplex loops in vivo. These loops, called t-loops, are formed by the strand invasion of the 3¢ tail of the telomere into the duplex telomeric repeat array. This structure is proposed to sequester the telomere terminus from cellular activities (DNA damage checkpoints, exonucleases, ligases) that could modify chromosome ends. In addition, t-loops might provide for a mechanism to regulate the access of telomerase to the telomere. We are currently testing these models.