Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements - PubMed (original) (raw)

Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements

J Yang et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The non-long terminal repeat (LTR) retrotransposon, R2, encodes a sequence-specific endonuclease responsible for its insertion at a unique site in the 28S rRNA genes of arthropods. Although most non-LTR retrotransposons encode an apurinic-like endonuclease upstream of a common reverse transcriptase domain, R2 and many other site-specific non-LTR elements do not (CRE1 and 2, SLACS, CZAR, Dong, R4). Sequence comparison of these site-specific elements has revealed that the region downstream of their reverse transcriptase domain is conserved and shares sequence features with various prokaryotic restriction endonucleases. In particular, these non-LTR elements have a Lys/Arg-Pro-Asp-X12-14aa-Asp/Glu motif known to lie near the scissile phosphodiester bonds in the protein-DNA complexes of restriction enzymes. Site-directed mutagenesis of the R2 protein was used to provide evidence that this motif is also part of the active site of the endonuclease encoded by this element. Mutations of this motif eliminate both DNA-cleavage activities of the R2 protein: first-strand cleavage in which the exposed 3' end is used to prime reverse transcription of the RNA template and second-strand cleavage, which occurs after reverse transcription. The general organization of the R2 protein appears similar to the type IIS restriction enzyme, FokI, in which specific DNA binding is controlled by a separate domain located amino terminal to the cleavage domain. Previous phylogenetic analysis of their reverse transcriptase domains has indicated that the non-LTR elements identified here as containing restriction-like endonucleases are the oldest lineages of non-LTR elements, suggesting a scenario for the evolution of non-LTR elements.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Identification of the putative endonuclease domain in R2 and other site-specific non-LTR elements. (A) Schematic diagram of the R2 ORF from B. mori and its comparison to the C-terminal ends of other site-specific non-LTR retrotransposable elements. Shaded regions in R2 indicate the RT domain, putative zinc finger (CCHH), and c-myb-like DNA-binding motifs. Carboxyl terminal to the RT domain in all elements is a putative zinc finger motif (CCHC) and a motif (PD..D) with similarity to restriction enzymes. (B) Sequence comparison of the putative endonuclease domain of nine R2 elements from diverse arthropods (9) with those of other sequence-specific non-LTR elements and with some restriction endonucleases. The number of amino acid residues between the conserved motifs is given in parentheses. Highly conserved residues are shown in shaded boxes, with the three charged residues that are part of the active site of the restriction enzymes also bolded. The region between these conserved residues assumes a β-turn in restriction enzymes (24, 25). The large number of charged residues in these β-turns are indicated as light gray.

Figure 2

Figure 2

Enzymatic activity of the R2 protein mutations. (A) TPRT assay by using a 110-bp 5′ end-labeled target. The two strands of the target DNA are represented by the straight lines, with the labeled 5′ ends noted with an asterisk. The cDNA made in the reaction is indicated by a straight line, and the RNA template is indicated by a dashed line. The 283-nt RNA template contains the sequence of the 3′ untranslated region of the silkmoth R2 element. In the absence of TPRT, 60- and 48-nt labeled DNA fragments are detected on a denaturing gel. If TPRT occurs, a 343-nt fragment also is generated. (B) Autoradiography of the TPRT reaction run on an 8% denaturing polyacrylamide gel. For each reaction, 15 ng (200 fmol) of end-labeled DNA and 4 ng (30 fmol) of protein were incubated for 30 min. Lanes: 1, no protein; 2, wild-type R2 protein; 3, PA..D mutation; 4, PE..D mutation; and 5, YAYD mutation. Numbers to the right indicate the lengths of the observed DNA products.

Figure 3

Figure 3

Endonuclease mutants can conduct the TPRT reaction on prenicked DNA substrates. (A) Schematic diagram summarizing the substrates and products of the TPRT reactions. The two strands of the DNA substrate are indicated by thin lines; the cDNA synthesized during the reaction is indicated with a thicker line and the RNA template is indicated with a wavy line. (B) Autoradiographs of the reaction products with the PA..D mutation (Left) and PE..D mutation (Right) separated on 1% agarose gels. Only plasmids that have undergone the TPRT reaction can be seen in these autoradiograms. For each reaction, 0.25 μg supercoiled or prenicked plasmid DNA was incubated with the R2 protein as in Fig. 2, except that 2 μCi [α-32P]dATP was added to each reaction. Lanes: 1 and 5, no protein controls; 2–4, 4, 8, and 16 ng of the mutant protein incubated with the supercoiled target; 6–8, 4, 8, and 16 ng of the mutant protein incubated with the prenicked target; and 9, 16 ng of wild-type protein incubated with supercoiled DNA.

Figure 4

Figure 4

Complementation of the endonuclease and RT mutations. Assays were performed as diagrammed in Fig. 2_A_, by using the 110-bp end-labeled DNA target. Lanes: 1, no protein; 2, 4 ng PE..D mutation; 3, 4 ng PE..D and 4 ng YAYD mutations; and 4, 4 ng YAYD mutation. Neither the PE..D or YAYD mutant alone can use the DNA target to prime reverse transcription of the 283-nt R2 RNA; however, a mixture of the two proteins is capable. Twice the total level of protein was added in lane 3 compared with the wild-type lane in Fig. 2 to enable the formation of an equivalent amount of an active heterodimer of PE..D and YAYD compared with a wild-type dimer. We have no direct evidence, however, that such a heterodimer is formed.

Figure 5

Figure 5

Comparison of the ORFs encoded by group II introns and non-LTR retrotransposable elements. The group II intron shown is the ltrB intron of Lactococcus lactis (37). The protein domains shared by other group II introns are shaded and are similar to those identified previously (41), except that the domains referred to as Z and X in that study are shown here as part of the RT domain (see ref. 8). The putative endonuclease domain of the group II introns is identified as HNH (38, 39). In the case of the non-LTR retrotransposons, schematic diagrams of the R2, L1, and Jockey elements are shown as representatives of the major non-LTR structures found to date. Other major lineages of non-LTR elements with these basic structures are listed within the parentheses (8). The CCHH, c-myb, CCHC, and PD..D domains of the R2 elements are described in Fig. 1. The AP-like endonuclease domain identified at the amino-terminal end of L1 and Jockey elements is labeled APE. Elements with structures similar to L1 contain a CCHC domain downstream of their RT domain; thus, this region is likely to be involved in DNA binding. Arrows represent the likely path of non-LTR evolution in eukaryotes based on the phylogeny of their RT domains (8).

Similar articles

Cited by

References

    1. Eickbush T H. In: The Evolutionary Biology of Viruses. Morse S S, editor. New York: Raven; 1994. pp. 121–157.
    1. Whitcomb J M, Hughes S H. Annu Rev Cell Biol. 1992;8:275–306. - PubMed
    1. Mizuuchi K. Annu Rev Biochem. 1992;61:1011–1051. - PubMed
    1. Luan D D, Korman M H, Jakubczak J L, Eickbush T H. Cell. 1993;72:595–605. - PubMed
    1. Martin F, Maranon C, Olivares M, Alonso C, Lopez M C. J Mol Biol. 1995;247:49–59. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources