Telomeres, the natural ends of linear eukaryotic chromosomes, are essential for cell viability and genome integrity1
. In most organisms, telomeric DNA consists of short repetitive sequences that terminates in 3’ single-stranded overhangs. Both the double stranded repeats and the 3’ overhangs of mammalian telomeres are bound by shelterin, a six-protein complex that exclusively associates with telomeres and protects chromosome ends from aberrant DNA repair activities2,3
. Telomeric proteins have undergone a rapid rate of change during evolution4
. Notably, repressor activator protein 1 (RAP1) is the only telomere protein that is conserved from budding and fission yeast to mammals. RAP1 contains a BRCT domain, one or two Myb domains, and an RCT (R
erminus) domain (). Despite this relatively conserved multi-domain architecture, RAP1 proteins in different organisms appear to have acquired diversified functions during evolution.
Figure 1 Structure of the human TRF2RBM-RAP1RCT complex. (a) Domain organization of the TRF2 and RAP1 polypeptide chains. In TRF2, the N-terminal basic region is colored in blue, the C-terminal Myb domain in magenta, the TRFH domain in orange, the RAP1-binding (more ...)
In mammalian cells, RAP1 is the least well-understood component of shelterin. RAP1 does not directly bind to telomeric DNA. Instead, it is recruited to telomeres through interaction between its C-terminal RCT domain and TRF2, another shelterin protein that binds to the duplex region of telomeres5
. TRF2 is essential in telomere end protection, since removal of TRF2 from telomeres initiates a potent DNA damage response (DDR) that activates ATM and the non-homologous end joining (NHEJ) pathway, resulting in massive end-to-end chromosome fusions6–10
. Recent studies suggested that the TRF2-RAP1 subcomplex is sufficient to suppress NHEJ both in vitro
and in vivo11,12
. Since the stability of mammalian RAP1 is dependent upon its interaction with TRF26
, it remains unclear whether TRF2, RAP1 or both proteins are required to protect telomeres.
Budding yeast Saccharomyces cerevisiae
Rap1) was discovered as a positive transcriptional regulator of genes for multiple growth-related genes such as the ribosomal protein genes13
. Later studies revealed that Sc
Rap1 is the major double-stranded telomeric repeat-binding protein in S. cerevisiae
and plays essential roles in telomere length regulation, subtelomeric gene silencing and chromosome end protection14
. While the central two Myb domains are responsible for the DNA binding activity of Sc
, the C-terminal RCT domain mediates chromatin recruitment of two sets of proteins, the Sir proteins (Sir3 and Sir4) for transcriptional silencing16
and the Rif protein (Rif1 and Rif2) for telomere length regulation17,18
. Although the crystal structure of Sc
is available (Feeser and Wolberger, 2008), how this domain recruits the Sir and the Rif proteins to telomeres still remains unknown.
Fission yeast Schizosaccharomyces pombe
Rap1) was identified based on its limited sequence similarity to Sc
. Like mammalian RAP1 but unlike budding yeast Sc
Rap1, fission yeast Sp
Rap1 lacks DNA binding activity and was believed to localizes to telomeres via interactions with Taz1, an ortholog of mammalian TRF1 and TRF219,20
. Deletion of fission yeast rap1
results in chromosome end-to-end fusions, telomere elongation, and derepression of telomere silencing, phenotypes reminiscent of those observed in taz1
Δ cells, suggesting a close relationship between Sp
Rap1 and Taz119,20
. However, Sp
Rap1 lacks an obvious RCT domain. Therefore, how Sp
Rap1 interacts Taz1 remains unclear.
To address these structural and functional questions of RAP1, we solved the three-dimensional crystal or solution structures of the RCT domains of human, fission yeast, and budding yeast RAP1 in complex with their respective binding partners, TRF2, Taz1 and Sir3. Our structurally focused biochemical, cellular, and genetic analyses revealed that RAP1 contains a remarkably conserved protein-protein interaction module that is utilized by both mammalian and fission yeast RAP1 proteins to interact with a telomeric double-stranded DNA binding protein for telomere regulation and protection. In contrast, budding yeast ScRap1 uses this module to recruit Sir3 to telomeres to mediate transcriptional silencing. Together, our results reveal that an evolutionarily conserved protein interaction module in RAP1 plays diverse roles at telomeres in different organisms.