Telomere Proteins


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 Telomere Proteins Background

Telomeres are complexes of DNA and protein that comprise the physical ends of linear chromosomes. The DNA is non-coding, repetitive sequence, and consists of tandem repeats that are rich in guanines and cytosines. The DNA terminus is not blunt, but instead terminates with an overhang on the 3' strand. The overhang is often referred to as the Goverhang because the sequence of the 3' strand is guanine-rich when compared to the complementary, 5' cytosine-rich strand. This overhang is important because it interacts with telomere proteins, helps the telomere form secondary structure, and serves as a substrate for the enzyme telomerase. Telomerase consists of both a reverse-transcriptase subunit (TERT) and an RNA component (TR) which together add repeats to the end of the DNA.

Telomeres serve multiple functions. These protein/DNA complexes serve a capping function to protect the chromosome end from being recognized as a double-strand break (DSB) and thus eliciting an aberrant DNA damage response. Also, telomeric DNA acts as a buffer for loss of sequence at the chromosome terminus by providing non-coding sequence that can be lost instead of critical coding regions. Sequence can be lost from the chromosome end for several reasons, including nuclease degradation and incomplete replication. A major obstacle facing cells with linear chromosomes is what comprises the "end-replication problem," which results from the inability of most DNA polymerases to completely replicate the 5' ends of the DNA. This issue occurs because all DNA polymerases synthesize DNA in a 5' to 3' direction and as a result, DNA is lost from the end of the lagging strand after the RNA primer is removed from the terminal Okazaki fragment. Telomeres solve this issue by providing non-coding sequence to be lost which protects important coding and regulatory regions and therefore genomic integrity.

Interactions between the DNA and protein components of telomeres, and in some organisms the formation of secondary structure, allow telomeres to hide the chromosome terminus from the DNA damage machinery. This protection is a critical function because if the end of the chromosome is seen as a double strand break the cell will try to "fix" the break through non-homologous end joining (NHEJ) which results in two chromosomes being ligated together, end-to-end. These fusion events are most often fatal for cells because during subsequent mitosis, chromosomes will not be able to segregate correctly resulting in chromosome breaks, aneuploidy, and genomic instability. In humans, telomere proteins also help the telomeric DNA form a secondary structure known as a T-loop in which the G-strand overhang is looped around and invades the double-stranded region. This effectively hides the overhang from degradation and the DNA damage response components, but also prevents it from being utilized as a substrate by telomerase to extend the telomere. Regulation of telomerase access to the telomere is another important role of telomere proteins and results in the modulation of telomere length. This regulation is achieved through direct and indirect interactions between telomeres and telomerase subunits. The regulatory function of telomere proteins is essential because telomere length is a limiting factor in the proliferative capacity of a cell and therefore access of telomerase to the telomere must be highly regulated.

There are various types of telomere proteins that have specific roles in telomere regulation. Proteins that bind to the G-overhang are essential for protection of the telomere end, and often they are involved in regulating telomerase access to the telomere. These proteins bind to the single-stranded overhang through domains called OB-folds. OB-fold motifs are DNA/RNA/oligosaccharide binding motifs. They can bind single-stranded nucleic acid in a sequence-independent manner as with the protein Replication Protein A (RPA), or they can have varying degrees of sequence specificity. Telomere proteins that bind the double-stranded region of the telomere do so through double-stranded DNA binding motifs known as MYB repeats. MYB motifs are characterized by clusters of α-helices and, like OB-folds, can bind to DNA with varying degrees of sequence specificity. There are additional proteins that do not bind to DNA directly but function through protein-protein interactions. These proteins can bridge the single-stranded and double-stranded binding components and also can interact with accessory proteins such as Werner's (Wrn) or Bloom (Blm) helicases. These non-DNA-binding telomere proteins are often equally essential to the protection and regulation of telomeres as their DNA-binding counterparts.

 

Mammalian telomere proteins and their functions

The telomere binding complex in humans is known as Shelterin because it "shelters" the telomere from degradation, erroneous repair, and inappropriate telomere length regulation. Shelterin is comprised of six proteins Pot1, Tpp1, Trf1, Trf2, Rap1, and Tin2. All except Rap1 are essential, although their functions vary widely.

In humans, the G-overhang binding protein is Pot1, for Protection of Telomeres 1. In these cells, the dominant role of Pot1 is the regulation of telomere length; however it also participates in the prevention of a DNA damage response via ATR activation. Pot1 contains single-strand nucleic acid binding motifs, known as OB-folds, which interact directly with the telomeric G-strand. The DNA damage response observed with depletion of Pot1 results in the accumulation of the single-strand binding protein RPA on the telomeric overhang. RPA binds to uncoated, single-stranded DNA with no sequence specificity and its accumulation can trigger the DNA damage response via ATR recruitment. ATR accumulation at the G-overhang indicates that Pot1 competes with RPA for binding to this single-stranded substrate. Interestingly, in-vitro, RPA has a higher, or at least a similar, affinity for the telomeric single-strand sequence as Pot1 suggesting that other factors are involved in the ability of Pot1 to protect the overhang from activation of ATR. Indeed it has been shown that the interaction of Pot1 with Tpp1, as well as their interaction with Tin2, is required for Pot1’s ability to antagonize RPA binding, and thereby for its protective functions at the overhang. Recent studies have also shown that secondary structure, in the form of a G-quadruplex, also enhances the ability of Pot1/Tpp1 to compete with RPA for overhang binding.