Affiliation:
1. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132
Abstract
ABSTRACT
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.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
Reference39 articles.
1. Ausubel
F. M.
Brent
R.
Kingston
R. E.
Moore
D. D.
Seidman
J. G.
Smith
J. A.
Struhl
K.
Current protocols in molecular biology.
1998
John Wiley & Sons Inc.
New York N.Y
2. LINEs and Alus—the polyA connection;Boeke J. D.;Nat. Genet.,1997
3. Capy
P.
Bazin
C.
Higuet
D.
Langin
T.
Dynamics and evolution of transposable elements.
1998
Landes Bioscience
Austin Tex
4. Christensen
S.
Ph.D. thesis.
1999
University of Utah
Salt Lake City
5. Targeting of human retrotransposon integration is directed by the specificity of the L1 endonuclease for regions of unusual DNA structure;Cost G. J.;Biochemistry,1998
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