Abstract
Accurate DNA replication requires a complex interplay of many regulatory proteins at replication origins. The CMG (Cdc45·Mcm2-7·GINS) complex, which is composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the heterohexameric origin recognition complex (ORC) and functions as a replicative helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. However, unlike archaea, trypanosomes possess components of the eukaryote-like CMG complex, but whether they form an active helicase complex, associate with the ORC, and regulate DNA replication remains unknown. Here, we demonstrated that the CMG complex is formed in vivo in trypanosomes and that Mcm2-7 helicase activity is activated by the association with Cdc45 and the GINS complex in vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, indicating that nuclear exclusion of Cdc45 constitutes one mechanism for preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 associate with Mcm2-7 via interactions with Mcm3. All together, we identified the Cdc45·Mcm2-7·GINS complex as the replicative helicase that interacts with two Orc1-like proteins in the unusual origin recognition complex in trypanosomes.
Highlights
After the pre-replicative complex (pre-RC) is assembled on replication origins, initiation of DNA replication is triggered by S-phase cyclin-dependent kinase (S-CDK) and Dbf4-dependent kinase Cdc7 [3]
We identified a novel Orc1-like protein, Orc1b, and established its interactions with Orc1/Cdc6 and Mcm3. These findings identified the Cdc451⁄7Mcm2–71⁄7GINS complex as an essential replicative helicase and an unusual origin recognition complex containing two Orc1-like proteins, Orc1/Cdc6 and Orc1b, in trypanosomes
We identified the Cdc451⁄7Mcm2–71⁄7GINS complex as the replicative helicase and an unusual origin recognition complex containing two Orc1-like proteins in trypanosomes
Summary
Trypanosome Cell Culture—The procyclic form of T. brucei strain 427 was cultured at 27 °C in SDM-79 medium supplemented with 10% fetal bovine serum (Atlanta Biologicals, Inc). Fluorescence Microscopy and Immunofluorescence Microscopy—Procyclic cells expressing endogenously EYFP-tagged Mcm and GINS proteins were harvested by centrifugation at 2,000 rpm for 5 min, washed once in PBS, and fixed in 4% paraformaldehyde. Cells were treated with blocking buffer (1% BSA, 0.1% Triton X-100 in PBS) for 1 h at room temperature and incubated with FITC-conjugated anti-HA monoclonal antibody (Sigma-Aldrich) diluted in PBS containing 1% BSA. Cdc, Psf, Orc1/Cdc, and Orc1b were each cloned into the pC-3HA-Bla vector, and the resulting constructs were transfected into the pC-Mcm3-PTP-Neo cell line. Protein Stability Assay—Trypanosome cell lines expressing endogenously HA-tagged Cdc, Mcm, Orc1/Cdc, or Psf were incubated with 100 g/ml cycloheximide (Acros Organics), and time course samples were collected for Western blot analysis.
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