An Alternative DNA Endonuclease Activity is Associated with the LINE-1 ORF2-encoded Protein

Area of Science:

• Molecular Biology
• Genetics
• Genomics

Background:

• Long Interspersed Element-1 (L1) retrotransposons mobilize via target-site primed reverse transcription (TPRT) using their ORF2p.
• L1 retrotransposition can occur independently of the L1 endonuclease (EN) domain, utilizing existing DNA breaks.

Purpose of the Study:

• To investigate the mechanism of EN-independent L1 retrotransposition.
• To characterize a potential alternative endonuclease activity within L1 ORF2p.

Main Methods:

• Purification of recombinant full-length human L1 ORF2p (wild-type, EN-mutant, RT-mutant) from insect cells.
• Assay of endonuclease activity using modified DNA primers and L1 RNA templates.
• Detection of alt-EN activity in bacterially expressed L1 ORF2p and a group II intron-encoded protein.

Main Results:

• Wild-type and L1 EN-mutant ORF2p, but not L1 RT-mutant, possess an alternative endonuclease (alt-EN) activity.
• Alt-EN activity was detected in truncated L1 ORF2p and a related protein, suggesting it resides within the reverse transcriptase fold.
• Mutations in the RT active site abolished endonucleolytic cleavage, indicating a link between RT and alt-EN.

Conclusions:

• An alternative endonuclease (alt-EN) activity is associated with the L1 reverse transcriptase domain.
• This conserved alt-EN activity may have facilitated EN-independent retrotransposition in early retrotransposons before the evolution of a dedicated EN domain.