Effects of Limiting Homology at the Site of Intermolecular Recombinogenic Template Switching during Moloney Murine Leukemia Virus Replication

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Abstract
A Moloney murine leukemia virus-based single-replication-cycle assay was developed to study the effects of limiting the extent of template and primer strand complementarity on recombinogenic template switching. This system mimicked forced copy choice recombination in which nascent DNA transfers from the end of a donor template to an acceptor position on the other copackaged RNA. When acceptor target regions with different extents of complementarity to the transferring DNA were tested, efficient recombination occurred with as few as 14 complementary nucleotides. The frequencies of correct targeting, transfer-associated errors, mismatch extension, and transfer before reaching the end of the donor template were determined. All four molecular events occurred, with their proportions varying depending on the nature of acceptor/transferring DNA complementarity. When complementarity was severely limited, recombination was inefficient and most products resulted from aberrant second-strand transfer rather than from forced template switching between RNAs. Other classes of reverse transcription products, including some that resulted from template switching between virus and host sequences, were also observed when homology between the acceptor and donor was limited.