The Human Rap1 Protein Complex and Modulation of Telomere Length

Abstract

Proper maintenance of telomere length and structure is necessary for normal proliferation of mammalian cells. Mammalian telomere length is regulated by a number of proteins including human repressor activator protein (hRap1), a known association factor of TRF2. To further delineate hRap1 function and its associated proteins, we affinity-purified and identified the hRap1 protein complex through mass spectrometry analysis. In addition to TRF2, we found DNA repair proteins Rad50, Mre11, PARP1 (poly(ADP-ribose) polymerase), and Ku86/Ku70 to be in this telomeric complex. We demonstrated by deletional analysis that Rad-50/Mre-11 and Ku86 were recruited to hRap1 independent of TRF2. PARP1, however, most likely interacted with hRap1 through TRF2. Interestingly, knockdown of endogenous hRap1 expression by small hairpin interference RNA resulted in longer telomeres. In addition, overexpression of full-length and mutant hRap1 that lacked the BRCA1 C-terminal domain functioned as dominant negatives and extended telomeres. Deletion of a novel linker domain of hRap1 (residues 199 -223), however, abolished the dominant negative effect of hRap1 overexpression. These results indicate that hRap1 negatively regulates telomere length in vivo and suggest that the linker region of hRap1 may modulate the recruitment of negative regulators of telomere length.Telomere-binding proteins TRF1 and TRF2 play pivotal roles in telomere protection and maintenance in mammalian cells (1-5). Several proteins have been shown to associate with TRF1 and TRF2 (3,5,6). Recently, a novel telomere regulator human repressor activator protein (hRap1) 1 was identified as a protein that specifically interacts with TRF2 (7). hRap1 is the human homologue of yeast RAP1. In yeast, RAP1 is a negative regulator of telomere length as well as a regulator of transcription (for review, see Refs. 8 -13). The RAP1 mutants in Saccharomyces cerevisiae (scRAP1) and Schizosaccharomyces pombe (spRAP1) are defective in telomere length control and telomere position effects (14 -16). In human cells overexpression of hRap1 extends telomeres (7,17). It has yet to be fully determined, however, whether endogenous hRap1 is a negative or positive regulator of telomere length. scRAP1 contains two Myb domains and binds telomeric DNA (18,19). scRAP1 has been shown to recruit Rif1, Rif2, and Sir proteins to regulate telomere length, structure, and transcriptional silencing (11,20). Whether similar proteins, as found with yeast RAP1, are complexed with hRap1 remains unknown. In contrast, spRAP1 has only one Myb domain and is recruited to telomeres through Taz1, a yeast homologue of TRF2 (15). Similar to spRAP1, hRap1 also contains a single Myb domain without detectable DNA binding activity (7,21). In addition to the Myb domain, hRap1 has three putative protein-protein interaction domains; they are a BRCT domain, a coiled-coil domain, and a TRF2 interacting RCT domain (7). The function of the hRap1 BRCT and coiled-coil domains has yet to be described.TRF2 has been shown to associate with t...

Telosome, a Mammalian Telomere-associated Complex Formed by Multiple Telomeric Proteins

“…Interference of endogenous TRF2 activity by expressing dominant negative forms of TRF2 markedly increased the rate of telomere end-to-end fusions (12). Consistent with this role of TRF2, TRF2 forms a complex with RAP1 and associates with several proteins involved in DNA damage and repair responses, notably RAD50/MER11/NBS1, Ku86, and ERCC1/ XPF (13)(14)(15). These findings have pointed to distinct biological functions of TRF1 and TRF2.…”

“…Mutants-FLAG-tagged full-length hRap1 and various hRap1 mutants were cloned in the pBabe-puro retroviral vector as previously described (15). The retroviral wild type and mutant FLAG-tankyrase (TANK) vectors were a generous gift from Dr. Titia de Lange (23).…”

Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference

“…RAD50, MRE11 and NBS1 are present in TRF2 immunocomplexes (Zhu et al, 2000) via their interaction with RAP1 (O'Connor et al, 2004). RAD50 and MRE11 are located at telomeres throughout interphase, and are joined by NBS1 in S-phase (Zhu et al, 2000).…”

“…The functions of TRF1 and TRF2 are interconnected and integrated by TIN2 (Houghtaling et al, 2004;Kim et al, 2004;. Also, TRF2 binds RAP1 (Li et al, 2000), which recruits MRN complexes (Zhu et al, 2000;O'Connor et al, 2004). Functional impairment of any of these proteins by RNA interference or expression of a dominant negative protein has shown that all four proteins negatively regulate telomere length in telomerasepositive cells (van Steensel and de Lange, 1997;van Steensel et al, 1998;Li and de Lange, 2003;O'Connor et al, 2004;Ye and de Lange, 2004).…”

“…Also, TRF2 binds RAP1 (Li et al, 2000), which recruits MRN complexes (Zhu et al, 2000;O'Connor et al, 2004). Functional impairment of any of these proteins by RNA interference or expression of a dominant negative protein has shown that all four proteins negatively regulate telomere length in telomerasepositive cells (van Steensel and de Lange, 1997;van Steensel et al, 1998;Li and de Lange, 2003;O'Connor et al, 2004;Ye and de Lange, 2004). Although the functions of these proteins in ALT, including APB assembly, are largely unknown, all four proteins are present in APBs (Yeager et al, 1999;Wu et al, 2003;this study).…”