The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus...

Area of Science:

• Molecular Biology
• Biochemistry
• Archaea Research

Background:

• Archaeal minichromosome maintenance (MCM) helicase activity is enhanced by GINS.
• Eukaryotic CMG (Cdc45-MCM-GINS) complex includes Cdc45, homologous to bacterial RecJ.
• Archaea possess Cdc45/RecJ-like proteins with GINS-stimulated exonuclease activity.

Purpose of the Study:

• To identify and characterize the CMG-like complex in Thermococcus kodakarensis.
• To investigate the function of the GINS-associated nuclease (GAN) in T. kodakarensis.
• To determine the role of GAN in DNA replication and cellular viability.

Main Methods:

• Identification of the CMG-like complex in T. kodakarensis.
• In vitro assays to assess helicase and nuclease activities of GAN, GINS, and MCM.
• Gene disruption analysis (Δgan mutant) to evaluate GAN's essentiality and growth phenotypes.
• Complementation studies using a nuclease-deficient GAN variant.

Main Results:

• The GAN·GINS complex stimulates MCM helicase activity in vitro.
• MCM does not affect the nuclease activity of GAN.
• GAN is non-essential for viability, but its absence (Δgan mutant) impairs growth at high temperatures (93°C) and causes growth retardation at optimal temperatures (85°C).
• Complementation with nuclease-deficient GAN restores growth, indicating the nuclease activity is unrelated to the observed defects.

Conclusions:

• The CMG-like complex, including GAN, is important for efficient replication fork progression in archaea.
• GAN is essential for optimal growth, particularly at high temperatures.
• The growth defects in Δgan mutants are linked to GAN's role in replication stability, not its nuclease activity.