Cooperation between two modes for DNA replication initiation in the archaeon Thermococcus barophilus.

Area of Science:

• Microbiology and Molecular Biology
• Genetics and Genomics
• Biochemistry

Background:

• Genomic DNA replication mechanisms in Archaea are not fully understood, particularly the role of replication origins (oriC).
• Some archaea can grow without a canonical origin of replication, suggesting alternative initiation pathways like recombination-dependent replication (RDR).
• The recombinase RadA is implicated in RDR, but its precise role and the interplay with oriC in natural growth conditions require further investigation.

Purpose of the Study:

• To investigate the dynamics of DNA replication initiation in Thermococcus barophilus throughout different growth phases.
• To elucidate the interplay between oriC utilization and recombination-dependent replication (RDR) in initiating DNA replication.
• To confirm the essentiality of RadA in RDR and its role in replication initiation.

Main Methods:

• Deep sequencing techniques were employed to analyze DNA replication patterns.
• Genetic manipulation, including the creation of a RadA knockdown mutant strain, was used to assess gene function.
• Analysis of ploidy in oriC and radA mutant strains was performed to understand its impact on cell survival.

Main Results:

• The utilization of oriC in T. barophilus diminishes from lag to mid-log phase and increases in the stationary phase.
• RadA was confirmed to be essential for growth, and its pivotal role in RDR was demonstrated for the first time using a knockdown mutant.
• DNA replication initiation relies on a combination of oriC and RDR, with their relative contributions varying across growth phases.

Conclusions:

• Thermococcus barophilus exhibits a flexible DNA replication initiation strategy, tightly integrating oriC utilization and homologous recombination.
• The study demonstrates that physiological state significantly influences DNA replication initiation, providing insights into environmental sensing impacts.
• RadA is essential for replication initiation in T. barophilus, particularly through RDR during log phase growth.