Intein splicing efficiency and RadA levels can control the mode of archaeal DNA replication

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Inteins are mobile genetic elements that excise themselves from precursor proteins via protein splicing.
• While in vitro studies suggest inteins regulate protein function, in vivo evidence in native hosts is lacking.
• Inteins are known to interrupt proteins involved in DNA replication, recombination, and repair.

Purpose of the Study:

• To investigate the in vivo role of intein splicing as a regulatory mechanism in the model archaeon Thermococcus kodakarensis.
• To determine the physiological and replication impacts of inhibiting intein splicing, specifically for the recombinase RadA.

Main Methods:

• Genetic manipulation of Thermococcus kodakarensis to inhibit intein splicing.
• Assessing host physiology, including growth rates and DNA damage sensitivity.
• Analyzing DNA replication modes using molecular biology techniques.

Main Results:

• Decreased intein splicing efficiency of RadA resulted in significant physiological consequences.
• Observed a general growth defect and increased sensitivity to DNA damage in the host organism.
• Identified a switch in DNA replication strategy from recombination-dependent to origin-dependent replication.

Conclusions:

• Intein splicing of RadA acts as a critical regulatory mechanism in vivo.
• Inhibition of intein splicing impacts fundamental cellular processes like DNA replication and repair.
• This study provides the first in vivo evidence for intein-mediated regulation in a native archaeal host.