Structural basis for the evolution of a domesticated group II intron-like reverse transcriptase to function in host...

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• A domesticated bacterial group II intron-like reverse transcriptase (G2L4 RT) was previously shown to function in double-strand break repair (DSBR) via microhomology-mediated end joining (MMEJ).
• Mobile group II intron-encoded RTs possess basal DSBR activity, utilizing structural features common to non-LTR-retroelement RTs.

Purpose of the Study:

• To determine the structural basis of G2L4 RT's function in DSBR.
• To elucidate novel structural adaptations that optimize G2L4 RT's cellular role in DSBR.

Main Methods:

• X-ray crystallography was used to determine the apoenzyme structure of G2L4 RT.
• Snap-back DNA synthesis structures of G2L4 RT were determined to understand substrate interaction.

Main Results:

• A unique RT3a structure was identified, stabilizing the apoenzyme in an inactive state until substrate binding.
• A longer N-terminal extension/RT0-loop and a modified active site were found to favor strand annealing.
• A conserved dimer interface facilitates G2L4 RT homodimer localization to DSBR sites, positioning monomers for MMEJ.

Conclusions:

• G2L4 RT possesses unique structural adaptations optimizing its cellular function in DSBR.
• These findings reveal the evolution of a non-LTR-retroelement RT for a dedicated cellular role.
• The study suggests potential for optimizing RTs for genome engineering applications.