Coordinated RNA- and protein-templated synthesis of double-stranded DNA by a dual reverse transcriptase immune system

Area of Science:

• Molecular Biology
• Microbial Immunity
• Enzymology

Background:

• Defense-associated reverse transcriptase (DRT) systems are crucial for antiviral immunity.
• DRTs utilize distinct cDNA synthesis modes: Class I (untemplated) and Class II (RNA-templated).
• The precise mechanisms by which DRTs drive antiviral defense are not fully understood.

Purpose of the Study:

• To elucidate the unprecedented mechanism of DRT3 immunity.
• To understand how Class I and Class II DRT enzymes cooperate in antiviral defense.
• To investigate the role of host recombination machinery in regulating DRT3 activity.

Main Methods:

• Utilized Cryo-electron microscopy to determine the structure of DRT3b.
• Investigated DRT3a and DRT3b enzymatic activities with different RNA templates.
• Assessed the toxicity of DRT3 systems in E. coli RecBCD-deficient strains.
• Examined the effect of phage Gam on DRT3-mediated abortive infection.

Main Results:

• DRT3 employs both Class I and Class II reverse transcriptases to produce self-complementary dsDNA.
• DRT3a uses an RNA template for poly-(dTdG) synthesis, while DRT3b synthesizes poly-(dCdA) repeats via amino acid-templated polymerization.
• DRT3 systems are toxic in E. coli lacking RecBCD, indicating host recombination limits dsDNA accumulation.
• The phage inhibitor Gam triggers DRT3-mediated abortive infection.

Conclusions:

• Two polymerases with distinct templating strategies cooperate to generate complementary DNA for antiviral defense.
• DRT3b utilizes a unique amino acid-templated mechanism for dinucleotide repeat synthesis.
• Host RecBCD recombination machinery plays a critical role in controlling DRT3-mediated dsDNA levels and bacterial immunity.