Chromosome ends, or telomeres, consist of repetitive DNA sequences and a plethora of protective proteins that are crucial for chromosome stability. Aberrations in either the DNA or the protein structures in this complex assembly lead to chromosome re-arrangements. The DNA repeat units are rich in G residues on the 3'-OH-bearing strand, which forms a single stranded overhang referred to as the G-tail. Shortening of telomere DNA occurs in many cells types due to incomplete end replication. This process can be counter-balanced by telomerase and DNA polymerase α, which synthesize the telomere ‘G-‘ and ‘C-strand', respectively. Abnormal telomere loss (due e.g., to telomerase deficiency) has been established as the cause of numerous diseases characterized by defective tissue renewal. Moreover, telomere dysfunction and telomerase up-regulation is known to contribute to the initiation and progression of human cancers.
My laboratory studies the mechanisms and regulations of telomere DNA synthesis with the ultimate aim of manipulating the relevant pathways for the treatment of cancers and age-related diseases. We utilize a variety of standard and unorthodox fungal model systems to dissect the mechanisms of key players in telomere DNA synthesis pathways. The main targets of our investigation are telomerase, DNA polymerase α, and the Cdc13-Stn1-Ten1 or CST complex.
• Telomerase: a special reverse transcriptase that is subject to complex regulation.
The extension of telomere G-strand is executed by telomerase, which contains two core components: an RNA that provides the template (TER), and a protein that synthesizes the repeats (TERT). Telomerase binds to the telomere G-tail through both protein-DNA and RNA-DNA interactions, and then catalyzes the extension of G-tail through reverse transcription of the RNA template. Unique among reverse transcriptases, telomerase is able to copy iteratively the template region of the RNA, thus adding multiple telomere repeats onto the G-strand without dissociation. This property (known as repeat addition processivity) requires an "anchor site" in telomerase that interacts with the 5' region of telomeric DNA during the "translocation" reaction. We and others have mapped anchor site to the conserved TERT essential N-terminal (TEN) domain and shown that this unique telomerase domain is regulated by a conserved OB fold protein (named TPP1 in humans and Est3 in budding yeast). A special current focus is to understand how the interaction between Est3 and TEN domain triggers the activation of telomerase.
• DNA polymerase α and telomere C-strand synthesis: a new trick for a classic DNA polymerase.
Because telomerase mediates the extension of only the G-strand, another activity is required to "fill-in" the telomere C-strand. This activity is believed to reside in the DNA Polymerase α-primase complex. Pol α-mediated C-strand synthesis has received far less attention than telomerase-mediated G-strand extension, but is no less important. Indeed, the distance between the 5' end of the C-strand and 3' end of G-strand controls the amount of telomere loss in the ensuing cell cycle. Thus, factors that determine the initiation property of Pol α-primase complex have a strong impact on telomere maintenance. We recently isolated the Pol α-primase complex from C. glabrata, and reconstituted the synthesis of telomere C-strand in vitro using model G-strand templates. Our system offers great opportunities for dissecting the molecular mechanisms of C-strand synthesis and its regulation.
• The Cdc13-Stn1-Ten1 complex: the G-tail binding complex that regulates both G- and C-strand synthesis.
A variety of G-tail binding proteins have been identified in different organisms and shown to mediate critical functions in telomere protection and maintenance. An especially important and well-conserved complex is the Cdc13-Stn1-Ten1 or CST complex. Initially identified in and thought to be confined to budding yeast, this complex is now known to be extremely widespread. Moreover, the CST complex has been shown to regulate both telomerase and Pol α activities in diverse organisms. Mutations in subunits of this complex engender telomere aberrations such as accumulation of long G-tails and abnormal recombination. Indeed, mutations in a human CST subunit (CTC1) was recently shown to be responsible for a complex disease named Coates plus.
The investigation of CST mechanisms has been hampered by the inability to express and purify adequate quantities of the complex. By screening multiple CST homologues for ease of expression and reconstitution, we succeeded in reconstituting and purifying the full C. glabrata (Cg) CST complex. We showed that the purified complex binds Cg Pol α and stimulates its activity in vitro, thus establishing a system for a detailed mechanistic and functional investigation. Current efforts are geared toward understanding the molecular basis of CST-Pol α interaction, and pinpointing the steps in the Pol α-primase reaction stimulated by CST.