Finally, our biochemical experiments demonstrate that template switching by the group II intron RT from the 59 end of the intron RNA directly to the 39 end of the upstream DNA exon is a potential alternate mechanism for cDNA attachment during linear intron RNA retrohoming. Although we found previously that the Ll.LtrB RT has low DNA-dependent DNA polymerase activity in vitro [21], we find here that it template switches and copies 59-exon DNA templates as well as 59-exon RNA templates (Figure 6), possibly reflecting that reverse transcription favors a conformation of the enzyme that can initiate more efficiently on DNA templates. In many cases, the template-switching junctions to DNA or RNA templates are seamless, but some have a small number of extra nucleotide residues, predominantly A-residues (corresponding to T-residues in the top strand) that were added by the RT to the 39 end of the cDNA prior to the template switch. This pattern of extra nucleotide addition, which we found under both enzyme optimal and near-physiological conditions, differs from the majority of 59-integration junctions resulting from linear intron retrohoming in vivo, where the extra nucleotide residues do not show a similar bias and sometimes correspond to copies of neighboring DNA sequences. It remains possible, however, that template switching by the Ll.LtrB RT could give different junctions in vivo, and that template switching and extra nucleotide addition by this enzyme contributes to some retrohoming events. We note that the ability of the group II intron RT’s template switching activity to efficiently link sequences in two different templates could potentially be used to directly attach linker sequences containing primer-binding sites to the ends of cDNAs for cDNA cloning and sequencing applications. Materials and Methods D. melanogaster stocks Flies were raised in standard fly media at 22uC. The lig4169 mutant, obtained from Mitch McVey (Tufts University, Medford, MA), has a deletion that removes the start codon and most of the region encoding the ATPase and adenylation domains [31]. The ku707B2 and ku70Ex8 mutants were obtained from William Engels (University of Wisconsin, Madison, WI). The ku707B2 allele lacks 1,359 bp at the 39 end of the 2,393-bp gene, including most of the DNA and Ku80-interaction domains [43]. The ku70Ex8 allele lacks at least 1 kb, including all of exon 1 and the start codon [43]. The ku707B2/ku70Ex8 genotype, generated by crosses between trans- heterozygous ku707B2/ku70Ex8 parents, is the same as that used previously to study double-strand break repair pathways [39,43]. The mus308D2 stock [60] was obtained from the Drosophila Stock Center (Bloomington, IA). The mutation lies outside of the coding region and results in undetectable levels of PolQ protein expression [38]. To obtain transgenic flies harboring a lig4 rescue fragment, a 6- kb DNA segment containing the lig4 gene was amplified from w1118 genomic DNA by using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN), with primers Lig4 F1 BamHI (59-AAGAGGATCCAGTAGCTGTAGAAG- CAGCCAAC) and Lig4 R1 XhoI 59-AAGACTCGAGCAG- CAGTTCCTCCGACATGAAG). This PCR product was insert- ed between BamHI and XhoI sites of the P-element transformation vector pCaSpeR4 [61], and transgenic flies were produced by GenetiVision (Houston, TX). A transgene insertion on chromosome 2 was recombined with lig4169 to generate the fly stock used in P{lig4+} rescue experiments. Except for the trans-heterozygous ku707B2/ku70Ex8 embryos (see above), embryos used for microinjection were obtained from crosses between isogenic wild-type or homozygous mutant parents. Linear Group II Intron Retrohoming in Drosophila For all stocks, precellular blastoderm embryos were collected in egg laying chambers in under 40 min, microinjected with recipient plasmids and group II intron RNPs, and incubated for 1 h at 30uC prior to DNA extraction. Recombinant plasmids pACD5C, which was used for synthesis of lariat and linear intron Ll.LtrB intron RNAs, is a derivative intron-donor plasmid pACD4C with a T7 promoter sequence inserted in the sense orientation at the SalI site in intron DIV [25,62]. pBRR3-ltrB, the target plasmid for intron-integration assays, contains the Ll.LtrB intron homing site (ligated exon 1 and 2 sequences of the ltrB gene from positions 2178 upstream to +91 downstream of the intron-insertion site) cloned upstream of a promoterless tetR gene in an AmpR pBR322-based vector [63]. pIMP-1P, used for expression of the LtrA protein for RNP reconstitution, contains the LtrA ORF cloned downstream of a tac promoter and W10 Shine-Dalgarno sequence in the expression vector pCYB2 (New England BioLabs, Ipswich, MA) [9]. LtrA is expressed from this plasmid as a fusion protein with a C-terminal tag containing an intein-linked chitin-binding domain, enabling LtrA purification via a chitin-affinity column, followed by intein- cleavage [9]. pMAL-LtrA, used for expression of the LtrA protein for biochemical assays, contains the LtrA ORF [64] cloned down- stream of a tac promoter and W10 Shine-Dalgarno sequence between BamHI and HindIII of the protein-expression vector pMAL-c2t. The latter is a derivative of pMal-c2x (New England BioLabs, Ipswich MA) with a TEV protease-cleavage site in place of the factor Xa site [65]. LtrA is expressed from this plasmid with an N-terminal fusion to maltose-binding protein (MalE), enabling its purification via an amylose-affinity column, followed by TEV- protease cleavage to remove the tag (see below). Preparation of Ll.LtrB lariat and linear RNAs Ll.LtrB-DORF intron RNAs were transcribed from DNA templates generated by PCR of plasmid pACD5C with primers that append a phage T3 promoter sequence (underlined in sequences below) [26]. For the lariat precursor RNA, the PCR primers were pACD-T3 (59-GGAGTCTAGAAATTAACCCT- CACTAAAGGGAATTGTGAGCG) and NheIR (59-CTAG- CAGCACGCCATAGTGACTGGCG), and for linear intron RNA, the PCR primers were T3LIS-1G (59-AATTAACCCT- CACTAAAGTGCGCCCAGATAGGGTGTTAAGTCAAG) and HPLC-purified LtrB940a (59-GTGAAGTAGGGAGG- TACCGCCTTGTTC). The PCR products were purified by using the Wizard SV Gel and PCR Clean-up System (Promega), extracted with phenol-chloroform-isoamyl alcohol (phenol-CIA; 25:24:1 by volume), ethanol precipitated, and dissolved in nuclease-free water. In vitro transcription and the preparation of lariat and linear intron RNAs were as described [26]. Preparation of LtrA protein and RNPs The LtrA protein used for RNP reconstitution was expressed in E. coli BL21(DE3) from the intein-based expression vector pImp- 1P and purified via a chitin-affinity column and intein cleavage, as described [9], except that the column buffer contained 50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, and 0.1% NP-40. Ll.LtrB RNPs were reconstituted with the purified LtrA protein and in vitro-synthesized lariat or linear Ll.LtrB-DORF intron RNA, as described [26], except that the final RNP pellet was dissolved in 10 mM KCl, 5 mM MgCl2, and 40 mM HEPES, pH 8.0. The LtrA protein used in biochemical assays was expressed in E. coli BL21(DE3) from the plasmid pMAL-LtrA. Cells were grown in PLoS Genetics | www.plosgenetics.org 12 February 2012 | Volume 8 | Issue 2 | e1002534