Roles for ORC in M phase and S phase

Area of Science:

• Molecular Biology
• Cell Cycle Regulation
• Yeast Genetics

Background:

• The origin recognition complex (ORC) initiates DNA replication in Saccharomyces cerevisiae.
• ORC assembly and activation are tightly regulated during the cell cycle, occurring in late M and S phases.
• Specific ORC subunits, like Orc5p, play critical roles at distinct cell cycle transitions (G1/S and M phase).

Purpose of the Study:

• To investigate the cell cycle roles of the Orc5p subunit of the origin recognition complex (ORC).
• To characterize the phenotypes associated with temperature-sensitive mutations in ORC subunits, specifically orc5-1 and orc2 alleles.
• To determine the mechanism underlying dominant cell cycle arrest observed in certain ORC mutants.

Main Methods:

• Utilized temperature-sensitive yeast mutants (orc5-1) to study ORC function.
• Employed cell synchronization techniques (M phase block and release) to precisely time cell cycle arrests.
• Analyzed the dominance of mutant phenotypes by introducing wild-type Orc5p and assessing genetic interactions with orc2 alleles.

Main Results:

• Temperature-sensitive orc5-1 mutants exhibited distinct cell cycle arrest phenotypes depending on initial synchronization: early M phase in asynchronous cells and G1/S boundary in M-phase-synchronized cells.
• The G1/S arrest caused by orc5-1 was not rescued by adding wild-type Orc5p during G1.
• Mutations in orc2 and orc5 displayed a dominant negative effect, inhibiting cell cycle progression, which was localized to the nucleus.

Conclusions:

• Orc5p is essential for progression through both G1/S and early M phase in yeast.
• Mutant ORC complexes, particularly those involving Orc2p and Orc5p, can exert dominant inhibition on DNA replication initiation.
• The nucleus-localized dominant inhibition suggests that mutant ORC complexes interfere with the function of wild-type ORC on chromosomes, blocking replication initiation at functional origins.