Different Faces of the Human Telomeric Protein TPP1

Abstract

Eukaryotic cells must overcome two major biological problems associated with linear chromosomes: the end protection and the end replication problems. The end protection problem occurs when the natural ends of linear chromosomes are misrecognized as DNA breaks within the genome, eliciting a DNA damage response. To counteract this, chromosome ends are capped with of a repetitive DNA sequence called telomeric DNA. Telomeric DNA is coated by the protein complex shelterin, thereby sequestering it from DNA damage response and repair machinery. The end replication problem occurs because replicative DNA polymerases are unable to catalyze the synthesis of DNA at the extreme ends of chromosomes, leading to the gradual loss of DNA during every replication cycle. The ribonucleoprotein complex telomerase helps compensate for this end attrition by catalyzing the addition of telomeric repeats to chromosome ends, enabling somatic and germ stem cells to replicate throughout the lifetime of a species. Together, shelterin and telomerase solve both problems associated with linear chromosomes. TPP1 is the only shelterin protein known to play a dual role at chromosome ends. Not only does it help protect telomeres from erroneous DNA damage response and repair machinery, but it also recruits and facilitates processivity of telomerase. My doctoral studies have focused on how TPP1 interacts with telomerase and other shelterin components to mediate its vital functions in somatic and germ cells. I demonstrate the importance of five residues at the N-terminus of the oligosaccharide/oligonucleotide binding (OB) domain (NOB) of TPP1 in telomerase recruitment to the telomere, telomerase stimulation by TPP1, and telomere lengthening in cells. Together with the previously discovered TEL patch, we have now defined an elaborate surface on TPP1 that we predict binds telomerase. Additionally, I describe the expression and separation-of-function of two isoforms of human TPP1. One, described here as TPP1-L, is annotated as a 544 amino acid protein initiating at methionine one. The other, referred to here as TPP1-S, is related to the rodent TPP1 and initiates at methionine 87. While both TPP1-S and TPP1-L recruit telomerase to the telomere, only TPP1-S activates telomerase for efficient telomere elongation. TPP1-S is the predominant isoform in somatic cells, but TPP1-L is the major isoform in differentiated male germ cells. I found that telomerase expression persists in germ cells, suggesting that TPP1-L may function to restrain telomerase in this context. TPP1 is not only important in end replication, but it also plays an essential role in end protection by interacting with its shelterin partners POT1 and TIN2. To understand how each of these interactions contributes to end protection, I conducted a homology assisted site-directed mutagenesis screen to map the POT1 and TIN2 interaction surfaces on TPP1. I find that the TPP1-TIN2 surface is larger than previously appreciated and is important for TIN2 mediated cooperativity of TPP1 and TRF2. Together these studies serve to better define how TPP1 functions in human cells to protect the integrity of chromosome ends.