the lig4 and polQ mutants, indicating that other DNA ligases and polymerases can serve as backups that perform the same functions at lower efficiency. Biochemical experiments show that another possible Lig4-independent mechanism, template switching by the group II intron RT from the 59 end of the intron RNA directly to the upstream exon DNA, is possible but gives junctions differing from the majority of those in vivo. It seems likely that the mechanism elucidated here involving host DNA ligases and repair polymerases is also used for linear intron RNA retrohoming in Xenopus laevis, where we observed similar 59-integration junctions [25], and more generally, in other eukaryotes, including mammalian cells, where it could have implications for group II intron-based gene targeting. This mechanism also provides a possible means for proliferation of non-branching group II introns in prokaryotes, some of which encode a Ku homolog and ATP-dependent DNA ligases along with DNA repair polymerases and use them in NHEJ pathways related to those of higher organisms [49]. Additionally, features of this mechanism, including the use of both Lig4-dependent and alt-EJ and the requirement for extra nucleotide addition to the cDNA end by a DNA repair polymerase, may be used to promote retrotransposition and mitigate DNA damage caused by LINE elements and other retrotransposons [33,50–52]. The involvement of Lig4 in linear intron RNA retrohoming in Drosophila is indicated by the findings that a lig4 mutation decreases the retrohoming efficiency of linear intron RNA by ,80% while having no effect on the retrohoming of lariat RNA, and that the decreased retrohoming efficiency of the linear intron in the mutant could be restored to wild-type levels by ectopic expression of Lig4 from a P-element insertion. Most if not all of the residual linear intron RNA retrohoming in the lig4 mutant appears to occur by using components of the alt-EJ pathway, as judged both by similar levels of activity and characteristics of the cDNA ligation junction, particularly patterns of extra nucleotide and the use of micro- homologies (see Results). In Drosophila, C-NHEJ and alt-EJ give generally similar double-strand break repair junctions, albeit with quantitative differences in the frequency of extra nucleotide addition in some assays [31,38,39], whereas in yeast or mammalian cells, alt-EJ junctions show increased deletion lengths and use of microhomologies [53–55]. The involvement of PolQ in linear intron RNA retrohoming is indicated by the findings that a PolQ mutation decreases the retrohoming efficiency by .99% and substantially decreases the frequency of 59-integration junctions having extra nucleotide residues (4–20% of junctions compared to 67% for wild-type Or-R assayed in parallel and .48% in all other strains; Figure 4 and Figure 5). The mutation also increases the frequency of junctions with long ($15 bp) 59-exon deletions and long ($5 nt) micro- homologies. The latter increase is particularly striking, as such long microhomologies were found at 56% of the total and 40% of the unique junction sequences from the polQ mutant, but were not found at junctions (170 total) from any of the other strains analyzed (Figure 4 and Figure 5). The residual extra nucleotide addition in the polQ mutant, which was also seen at 15–20% of junctions in end-joining assays [38,39], could be due to small amounts of the enzyme remaining in the mutant, which has an unidentified expression defect, or to an alternate DNA polymer- ase. The very strong decrease in linear intron RNA retrohoming efficiency in the polQ mutant (.99%) indicates that PolQ functions in both the Lig4-dependent and Lig4-independent retrohoming pathways. PolQ could potentially play at least two roles in linear intron RNA retrohoming. First, extra nucleotide addition to the 39 end of the cDNA by PolQ may be critical for generating microhomol- ogies that can base pair with the upstream exon to facilitate DNA Linear Group II Intron Retrohoming in Drosophila ligation. Second, PolQ contains a putative DNA helicase domain that could also contribute to retrohoming by promoting base pairing between microhomologies at the cDNA end and the upstream exon, either by annealing the cDNA end to comple- mentary exon sequences or by unwinding the exon DNA strands, making the top strand more accessible to base pairing [38]. The increased frequency of long 59-exon deletions in the polQ mutant may reflect a delay in cDNA attachment due to lack of suitable microhomologies and/or impaired annealing of complementary cDNA ends to the top strand. The striking increase in the frequency of long microhomologies at the 59 junctions in the polQ mutant (see above) indicates that an alternate annealing mechanism exists in the polQ mutants, but that it is more dependent upon longer microhomologies between exon and intron sequences than the PolQ-assisted mechanism. The function, if any, of Ku in linear intron RNA retrohoming is unclear. The finding that ku70 mutations moderately inhibit retrohoming of both linear and lariat intron RNA (54 and 33% inhibition, respectively) could reflect either that Ku contributes to both pathways or that Ku mutations affect one or both pathways indirectly. The Ku protein interacts with a stem-loop region of the RNA component of yeast and human telomerase [56–58], and it is possible that Ku may similarly bind to linear or lariat group II intron RNAs to protect them from degradation and/or recruit other DNA repair enzymes to the site. An alternate possibility is that Ku affects retrohoming efficiency indirectly by contributing to the repair of double-strand breaks induced by the intron RNP in the recipient plasmids. In yeast mitochondria, double-strand breaks resulting from abortive retrohoming events are substantially more frequent than completed integrations [59]. If not repaired correctly, such double-strand breaks could lead to loss of functional recipient plasmid target sites, which would appear as decreased retrohoming efficiencies in our assay. A similar indirect effect, involving repair of double-strand breaks in the recipient plasmid could also account for the moderate inhibitory effect of the polQ mutation on lariat intron RNA retrohoming. Lig4 is ordinarily recruited to DNA breaks by Ku, and D. melanogaster mutations in either lig4 or ku70 give similar decreases in NHEJ efficiency, suggesting that Lig4 acts exclusively in Ku- dependent NHEJ [41–43]. By contrast, we find that linear intron RNA retrohoming is more strongly inhibited by a lig4 mutation than by putative mutations in ku70 (82 and 54% inhibition, respectively). Even assuming that the inhibition by the ku70 mutations is a direct effect, these findings most simply suggest that a substantial proportion of linear intron RNA retrohoming events are promoted by Lig4 in the absence of Ku. Unlike a conventional double-strand break, the double-strand break formed during linear intron RNA retrohoming has an RNA attached to one of the DNA ends, and this difference could potentially affect the recruitment and use of NHEJ activities. We noted previously that group II intron RNPs bind to both the 59- and 39-exons during retrohoming, and such bridging of the ligation junction could impede access and decrease the need for Ku to cap the broken DNA ends [25]. Additionally, the attached intron RNA could contribute directly to the recruitment of NHEJ activities. The interaction of Ku with telomerase RNA noted above is thought to help recruit telomerase to double-strand breaks [57], and it is possible that a similar interaction between Ku and the attached group II intron RNA contributes to the recruitment of Lig4 for some linear intron RNA retrohoming events. More generally, such a mechanism involving the interaction of Ku with RNA could also be used by LINE elements and other retro- transposons to recruit Lig4 and other NHEJ activities for cDNA integration and repair of DNA breaks. PLoS Genetics | www.plosgenetics.org 11 February 2012 | Volume 8 | Issue 2 | e1002534