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The human LINE-1 reverse transcriptase:effect of deletions outside the common reverse transcriptase domain

A P Clements¹, M F Singer

¹Laboratory of Biochemistry, National Cancer Institute, Building 37, Room 4A-01, Bethesda, MD 20892, USA.

Nucleic Acids Research

|July 22, 1998

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The Z region of LINE-1 ORF2 protein is crucial for reverse transcriptase activity. Specific amino acid changes in this region, but not in the endonuclease or cysteine-rich domains, abolish enzyme function.

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

LINE-1 elements are retrotransposons essential for genome plasticity.
LINE-1 ORF2 protein possesses both endonuclease and reverse transcriptase activities.
Understanding ORF2 protein function is key to LINE-1 biology.

Purpose of the Study:

To investigate the functional domains of the human LINE-1 ORF2 protein.
To identify regions critical for reverse transcriptase activity.

Main Methods:

Heterologous expression of human LINE-1 ORF2 and its deletion derivatives in yeast.
Co-purification of ORF2 protein with reverse transcriptase activity.
Reverse transcriptase assays using synthetic polynucleotides.
Site-directed mutagenesis of conserved regions.

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Main Results:

ORF2 protein expressed in yeast exhibits reverse transcriptase activity.
Deletion of the N-terminal endonuclease domain retains partial activity.
Deletion of the C-terminal cysteine-rich motif has minimal impact on activity.
Deletions within the Z motif region, particularly near the polymerase domain, abolish activity.
Mutations in the conserved Z8 octapeptide sequence can inactivate the enzyme.

Conclusions:

The Z region, including the Z8 octapeptide, is essential for LINE-1 ORF2 reverse transcriptase activity.
The endonuclease and cysteine-rich domains are not strictly required for reverse transcriptase function.
These findings provide insights into the structure-function relationship of LINE-1 reverse transcriptase.