The human Rap1 protein complex and modulation of telomere length.
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.
O'Connor
Matthew S
MS
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Safari
Amin
A
Liu
Dan
D
Qin
Jun
J
Songyang
Zhou
Z
D034961:Antigens, Nuclear / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D017353:Gene Deletion; D005799:Genes, Dominant; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D009154:Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012190:Retroviridae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D016615:Telomere / Q000648:ultrastructure*; D013997:Time Factors; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C086930:RAD50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D011065:Poly(ADP-ribose) Polymerases; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae; C000606509:Xrcc6 protein, human; D020827:rap1 GTP-Binding Proteins; D000072200:Ku Autoantigen