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Molecular and Cellular Biology, February 2000, p. 1219-1226, Vol. 20, No. 4
Department of Biochemistry, University of
Utah School of Medicine, Salt Lake City, Utah 84132
Received 14 September 1999/Returned for modification 2 November
1999/Accepted 15 November 1999
Elements of the Tx1L family are non-long terminal repeat
retrotransposons (NLRs) that are dispersed in the genome of
Xenopus laevis. Essentially all genomic copies of Tx1L are
found inserted at a specific site within another family of transposable
elements (Tx1D). This suggests that Tx1L is a site-specific
retrotransposon. Like many (but not all) other NLRs, the
Xenopus element encodes an apparent endonuclease that is
related in sequence to the apurinic-apyrimidinic endonucleases that
participate in DNA repair. This enzyme is thought to introduce the
single-strand break in target DNA that initiates transposition by the
target-primed reverse transcription (TPRT) mechanism. To explore the
issue of target specificity more fully, we expressed the polypeptide
encoded by the endonuclease domain of open reading frame 2 from Tx1L
(Tx1L EN) and characterized its cleavage capabilities. This
endonuclease makes a specific nick in the bottom strand precisely at
one end of the presumed Tx1L target duplication. Because this activity
leaves a 5'-phosphate and 3'-hydroxyl at the nick, it has the location
and chemistry required to initiate new insertion events by TPRT. Tx1L
EN does not make a specific cut at a preferred target site for Tx1D
elements, ruling out the alternative possibility that the composite
Tx1L-Tx1D element moves as a unit under the control of functions
encoded by Tx1L. Further characterization revealed that the
endonuclease remains active for many hours at room temperature and that
it is capable of enzymatic turnover. Scanning substitution mutagenesis located the recognition site for Tx1L EN within 10 bp surrounding the
primary nick site. Implications of these features for natural transposition events are discussed.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Target Specificity of the Endonuclease from the
Xenopus laevis Non-Long Terminal Repeat
Retrotransposon, Tx1L

and
*
Corresponding author. Mailing address: Department
of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132. Phone: (801) 581-5977. Fax: (801) 581-7959. E-mail: carroll{at}medschool.med.utah.edu.
Present address: Department of Biology, University of Rochester,
Rochester, NY 14627.
Present address: Huntsman Cancer Institute, University of Utah
School of Medicine, Salt Lake City, UT 84132.
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