Nucleic Acids Research Advance Access published online on September 12, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn554
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Molecular Biology |
A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity
1Department of Cell and Developmental Biology and Program in Molecular Biology, 2Human Medical Genetics Program, University of Colorado School of Medicine, Aurora, CO 80045, 3Department of Physics and 4Center for Interdisciplinary Research on Complex Systems, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA
To whom correspondence should be addressed. Tel: +1 303 724 3467; Fax: +1 303 724 3420; Email: sandy.martin{at}ucdenver.edu Correspondence may also be addressed to Mark C. Williams. Tel: +1 617 373 7323; Fax: +1 617 373 2943; Email: mark{at}neu.edu
Received June 23, 2008. Revised July 30, 2008. Accepted August 14, 2008.
L1 is a ubiquitous interspersed repeated sequence in mammals that achieved its high copy number by autonomous retrotransposition. Individual L1 elements within a genome differ in sequence and retrotransposition activity. Retrotransposition requires two L1-encoded proteins, ORF1p and ORF2p. Chimeric elements were used to map a 15-fold difference in retrotransposition efficiency between two L1 variants from the mouse genome, TFC and TFspa, to a single amino acid substitution in ORF1p, D159H. The steady-state levels of L1 RNA and protein do not differ significantly between these two elements, yet new insertions are detected earlier and at higher frequency in TFC, indicating that it converts expressed L1 intermediates more effectively into new insertions. The two ORF1 proteins were purified and their nucleic acid binding and chaperone activities were examined in vitro. Although the RNA and DNA oligonucleotide binding affinities of these two ORF1 proteins were largely indistinguishable, D159 was significantly more effective as a nucleic acid chaperone than H159. These findings support a requirement for ORF1p nucleic acid chaperone activity at a late step during L1 retrotransposition, extend the region of ORF1p that is known to be critical for its functional interactions with nucleic acids, and enhance understanding of nucleic acid chaperone activity.