Semirandom DNA adducts regulate a filamentous defense-associated reverse transcriptase

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• Bacteriophage defense systems utilize non-genomic DNA synthesis for immunity.
• The mechanisms by which undefined DNA sequences confer antiphage defense remain unclear.

Purpose of the Study:

• To elucidate the structure and function of the DRT1 antiphage defense system.
• To understand how DRT1 synthesizes non-genomic DNA and regulates its activity.

Main Methods:

• Cryo-electron microscopy (cryo-EM) for structural determination.
• Biochemical assays to characterize DNA synthesis and enzyme activity.
• Analysis of bacteriophage escape mutants.

Main Results:

• DRT1 performs template-free, protein-primed DNA synthesis, generating semirandom DNA adducts.
• DNA synthesis activates the nitrilase domain, and adducts induce quiescent filament formation.
• Filamentous DRT1 features domain-swapped C termini forming pseudoknots, occluding active sites and causing dormancy.
• A T4 single-stranded DNA helicase is essential for DRT1 activity.

Conclusions:

• DRT1 functions as a minimal retron, integrating reverse transcriptase, effector, and non-genomic antitoxin DNA production.
• The self-assembly into dormant filaments is a key regulatory mechanism for DRT1 activity.
• Understanding DRT1 provides insights into novel bacteriophage defense strategies and DNA synthesis mechanisms.