Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase

Area of Science:

• Molecular Biology
• Cell Cycle Regulation
• DNA Replication

Background:

• DNA replication origin licensing ensures each DNA segment is replicated once per cell cycle.
• This process involves loading six Minichromosome Maintenance (MCM) proteins as double hexamers, forming prereplicative complexes (pre-RCs).
• Assembly requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1, all AAA+ ATPases, and ATP hydrolysis.

Purpose of the Study:

• To investigate the specific roles of ATP binding and hydrolysis by ORC, Cdc6, and MCM in pre-RC assembly.
• To clarify the function of ATP hydrolysis in releasing nonproductive intermediates and facilitating productive complex formation.

Main Methods:

• Utilized mutant proteins of ORC, Cdc6, and MCM defective in ATP hydrolysis or binding.
• Analyzed the effects of these mutations on origin licensing, intermediate release, and double hexamer formation.

Main Results:

• ORC and Cdc6 mutants deficient in ATP hydrolysis can still license origins.
• ATP hydrolysis by Cdc6 is necessary to release nonproductive licensing intermediates.
• ATP binding stabilizes the wild-type MCM hexamer.
• ATP binding and hydrolysis by MCM are essential for Cdt1 release and the formation of MCM double hexamers.

Conclusions:

• The function of ATP hydrolysis in pre-RC assembly is more nuanced than previously thought.
• Cdc6's ATP hydrolysis primarily serves to clear unproductive intermediates.
• MCM's ATP binding and hydrolysis are critical for the final steps of double hexamer formation and Cdt1 release, impacting overall licensing efficiency.