Regulation of DNA-replication origins during cell-cycle progression

Area of Science:

• Molecular Biology
• Cell Cycle Regulation
• DNA Replication

Background:

• Chromosome VI in Saccharomyces cerevisiae has nine DNA replication origins with varying initiation frequencies.
• These origins replicate sequentially during the S phase of the cell cycle.

Purpose of the Study:

• To investigate the link between multiple origin activation and S-phase progression regulation.
• To determine the effects of DNA damage (MMS) and checkpoint gene mutations (rad53) on origin activity and cell-cycle progression.

Main Methods:

• Two-dimensional gel electrophoresis to measure origin activity.
• Fluorescence-activated cell sorting to monitor cell-cycle progression.
• Utilizing methyl methane sulphonate (MMS) for DNA damage induction.
• Employing mutations in checkpoint genes, specifically rad53.

Main Results:

• MMS treatment slows S-phase and selectively blocks initiation from late replication origins.
• A rad53 mutation enhances late/inefficient origins and overrides MMS-induced initiation blocks.
• rad53 mutation causes a late origin to replicate earlier.
• orc2 mutations are also involved in suppressing late origins.

Conclusions:

• rad53 regulates the timing of late origin initiation during normal cell growth.
• rad53 acts as a surveillance mechanism, blocking late origin initiation in MMS-treated cells.
• rad53 plays a role in monitoring S-phase progression.