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Abstract
Background
Alu elements are active non-LTR retrotransposons that account for approximately 11
% of the human genome and are known to be not only the cause of genetic disease in
germinal cells, but have also been implicated on the development of B-cell lymphomas
suggesting they affect the genome of somatic cells as well. In the present study,
the molecular mechanisms of the inhibitory activity of human APOBEC3 (A3) family members
on Alu retrotransposition were investigated.
Materials and methods
A3 family proteins were tested in a neomycin-resistant (neor)-based retrotransposition
assay. Briefly, HeLa cells were cotransfected with a neor-Alu reporter vector, LINE-1
ORF2 vector and the respective A3 expression plasmids. Seventy-two hours post-transfection,
G418 was added for resistance selection, and after 14 days resultant G418-resistant
colonies were fixed, stained with crystal violet and counted. A series of APOBEC3G
(A3G) mutants bearing deletions in the N-terminus in multiples of 30 amino acids (aa)
were constructed to evaluate the responsible region. Likewise, dimerization and deamination
deficient A3G mutants were also created to examine the inhibitory effect of A3G on
Alu retrotransposition.
Results
The neor-based retrotransposition assay showed that all hA3 family proteins differentially
inhibited Alu retrotransposition. By the deletion analyses based on A3G that is the
most well-characterized A3 family member, the N-terminal 30 aa of A3G was found to
determine its inhibitory activity on Alu retrotransposition. Mutational analyses showed
that the inhibitory activity of A3G was independent of its deaminase activity, but
dependent on its dimerization, which was found to be affected by deletion in its N-terminal
30 aa. Importantly, the dimerization of A3G was also required for the inhibition of
LINE-1 retrotransposition.
Conclusions
The N-terminal 30 aa of A3G are essential for its inhibitory activity on Alu retrotransposition,
and this correlates with A3G homodimerization. The structural basis for the effect
of the N-terminal 30 aa region of A3G on its dimerization is currently under investigation.
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