Linear Group II Intron Retrohoming in Drosophila nucleotide addition may be critical for generating microhomolo- gies that enable annealing between the 39 end of the cDNA and the upstream exon DNA. Further, the strongly decreased frequency of linear intron RNA retrohoming in the polQ mutant indicates that PolQ functions in both the Lig4-dependent and Lig4-independent retrohoming pathways. The Ll.LtrB RT can template switch from the 59 end of linear intron RNA to the upstream exon Although the residual linear intron RNA retrohoming events in the lig4 mutant can be accounted for by Lig4-independent (alt-EJ) NHEJ, it remained possible that template switching of the RT from the 59 end of the intron RNA directly to the 39 end of the 59- exon DNA contributes to this process. Previous studies have shown that other non-LTR retroelement RTs are proficient at template switching directly to the 39 end of a template strand with little or no complementarity to the cDNA end and that these events can be accompanied by extra nucleotide addition at the junctions, as found for NHEJ [35,36,37,44]. To determine if a template-switching mechanism could be responsible for the manner of 59 junctions observed during linear intron retrohoming, we carried out biochemical assays using small artificial substrates that simulate the situation at the 59-integration junction just prior to completion of intron cDNA synthesis (Figure 6). The primary substrate consists of a 60-nt RNA template (Ll.LtrB RNA), whose 59 end corresponds to that of the Ll.LtrB intron, with a 45-nt DNA primer representing the nascent cDNA (primer c) annealed to its 39 end. The Ll.LtrB RT (LtrA) initiates Figure 6. Template switching of LtrA from the 59 end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 59 end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 59-32P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 mM) in reaction medium containing 450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30uC. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 59-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing (Figure 7) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 59-end labeled size markers (10-bp DNA ladder, Invitrogen). doi:10.1371/journal.pgen.1002534.g006 PLoS Genetics | www.plosgenetics.org 8 February 2012 | Volume 8 | Issue 2 | e1002534