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Background: R2 elements are a clade of early branching Long Interspersed Elements (LINEs). LINEs are retrotransposable elements whose replication can have profound effects on the genomes in which they reside. No crystal or EM structures exist for the reverse transcriptase (RT) and linker regions of LINEs. Results: Using limited proteolysis as a probe for globular domain structure, we show that the protein encoded by the Bombyx mori R2 element has two major globular domains: (1) a small globular domain consisting of the N-terminal zinc finger and Myb motifs, and (2) a large globular domain consisting of the RT, linker, and type II restriction-like endonuclease (RLE). Further digestion of the large globular domain occurred within the RT. Mapping these RT cleavages onto an updated model of the R2Bm RT indicated that the thumb of the RT was largely protected from proteolytic cleavage. The crystal structure of the large globular domain of Prp8, a eukaryotic splicing factor, was a major template used in building the R2Bm RT model, particularly the thumb region. The large fragment of Prp8 consists not only of a RT similar to R2Bm, but also an RLE and a linker connecting the two regions. The linker sequences adjacent to the RLE in LINEs and Prp8 share a set of two important α-helices and a (presumptive) knuckle/ββα structural motif that are closely associated with the thumb. The RLEs of LINEs and Prp8 share a unique catalytic core residue spacing as well as other key residues. Conclusions: The protein encoded by RLE LINEs consists of two major globular domains. The larger of the two globular domain contains the RT, linker, and RLE and is similar to the large fragment of the spliceosomal protein Prp8. The similarities are suggestive of possible common ancestry. [ABSTRACT FROM AUTHOR]

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5′ end of the R2 element. In the absence of this 5′ RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3′ untranslated region of the R2 transcript is present. In the presence of the 320-nt 5′ RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5′ RNA is removed from this subunit. We postulate that the removal of the 5′ RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction. [ABSTRACT FROM AUTHOR]

R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons. [ABSTRACT FROM AUTHOR]

We report an electrical scheme to detect specific DNA. Engineered hairpin probe DNA are immobilized on a silicon chip between gold nanoelectrodes. Hybridization of target DNA to the hairpin melts the stem nucleotides. Gold nanoparticle-conjugated universal reporter sequence detects the open hairpins by annealing to the exposed stem nucleotides. The gold nanoparticles increase charge conduction between the electrodes. Specifically, we report on a hairpin probe designed to detect a medically relevant mutant form of the K-ras oncogene. Direct current measurements show three orders of magnitude increase in conductivity for as low as 2 fmol of target molecules. [ABSTRACT FROM AUTHOR]

Linear chromosomes shorten in every round of replication. In Drosophila, telomere‐specialized long interspersed retrotransposable elements (LINEs) belonging to the jockey clade offset this shortening by forming head‐to‐tail arrays at Drosophila telomere ends. As such, these telomeric LINEs have been considered adaptive symbionts of the genome, protecting it from premature decay, particularly as Drosophila lacks a conventional telomerase holoenzyme. However, as reviewed here, recent work reveals a high degree of variation and turnover in the telomere‐specialized LINE lineages across Drosophila. There appears to be no absolute requirement for LINE activity to maintain telomeres in flies, hence the suggestion that the telomere‐specialized LINEs may instead be neutral or in conflict with the host, rather than adaptive. [ABSTRACT FROM AUTHOR]