Recombination products suggest the frequent occurrence of aberrant gene replacement in the moss Physcomitrella patens

In gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. Gene replacement in the moss Physcomitrella patens is extremely efficient, but often large amounts of additional DNA are integrated at the target locus. A detailed analysis of recombination junctions of PpCOL2 gene knockout mutants shows that the integrated DNA can be highly rearranged. Our data suggest that the replaced sequences were excised by HR and became integrated back into the genome by non-homologous end-joining (NHEJ). RAD51-mediated strand-invasion and subsequent strand-exchange is central to the two-end invasion pathway, the major gene replacement pathway in yeast. In this pathway, integration is initiated by the free ends of a single replacement vector-derived donor molecule which then integrates as an entity. Gene replacement in P. patens is entirely RAD51-dependent suggesting the existence of a pathway mechanistically similar to two-end invasion. However, invasion of the two ends does not seem to be stringently coordinated in P. patens. Actually, often only one fragment end became integrated by HR, or one-sided integration of two independent donor fragments occurred simultaneously leading to a double-strand break that is subsequently sealed by NHEJ and thus causes the observed rearrangements.