TRF2 recruits ORC through TRFH domain dimerization.
Telomeres are specialized chromatin structures that prevent the degradation and instability of the ends of linear chromosomes. While telomerase maintains long stretches of the telomeric repeat, the majority of telomeric DNA is duplicated by conventional DNA replication. A fundamental step in eukaryotic DNA replication involves chromatin binding of the origin recognition complex (ORC). In human cells, telomeric repeat binding factor 2 (TRF2) is thought to play a role in the recruitment of ORC onto telomeres. To better understand the mechanism of TRF2-mediated ORC recruitment, we utilized a lacO-LacI protein tethering system in U2OS cells and found that ectopically targeted TRF2, but not TRF1, can recruit ORC onto the lacO array. We further found that the TRF homology (TRFH) dimerization domain of TRF2, but not its mutant defective in dimerization, is sufficient for ORC and minichromosome maintenance (MCM) recruitment. Mutations impairing the dimerization also compromised ORC recruitment by full-length TRF2. Similar results were obtained using immunoprecipitation and GST pull-down assays. Together, these results suggest that dimerized TRF2 recruits ORC and stimulates pre-replication complex (pre-RC) formation at telomeres through the TRFH domain.
Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Mitsunori
M
Higa
Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Tatsunori
T
Kushiyama
Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Seiichiro
S
Kurashige
Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Daisuke
D
Kohmon
Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Kouki
K
Enokitani
Division of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Satoko
S
Iwahori
Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Nozomi
N
Sugimoto
Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: kyoshida@phar.kyushu-u.ac.jp.
Kazumasa
K
Yoshida
Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: mfujita@phar.kyushu-u.ac.jp.
Masatoshi
M
Fujita
D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D004261:DNA Replication; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D056968:Lac Repressors / Q000235:genetics / Q000378:metabolism; D064110:Minichromosome Maintenance Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D000072417:Protein Domains; D055503:Protein Multimerization; D015398:Signal Transduction; D016615:Telomere / Q000378:metabolism* / Q000648:ultrastructure; D035321:Telomeric Repeat Binding Protein 1 / Q000235:genetics / Q000378:metabolism; D035341:Telomeric Repeat Binding Protein 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002843:Chromatin; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D051738:Origin Recognition Complex; C504753:TERF2 protein, human; D035321:Telomeric Repeat Binding Protein 1; D035341:Telomeric Repeat Binding Protein 2; D064110:Minichromosome Maintenance Proteins
DNA replication; MCM; ORC; Pre-replication complex; TRF2; Telomere