Effects of identity minimization on Moloney murine leukemia virus template recognition and frequent tertiary template-directed insertions during nonhomologous recombination.

Homology requirements for Moloney murine leukemia virus recombination were addressed in this study by monitoring titer defects observed when acceptor/donor template identity lengths were systematically reduced. Recombination acceptors with at least 16 contiguous bases of donor template identity were recognized as efficiently as longer acceptors. In contrast, a sharp 1-log titer drop was observed for an acceptor of only 15 bases long, with an additional 1-log titer decline for an 8-base acceptor and further decreases for shorter acceptors. Eighty-three independent nonhomologous recombination products were sequenced to examine recombination template selection in the absence of significant sequence identity. These replication products contained a total of 152 nonhomologous crossover junctions. Forced copy choice models predict that forced nonhomologous recombination should result in DNA synthesis to the donor template's 5' end, followed by microidentity-guided acceptor template selection. However, only a single product displayed this structure. The majority of examined nonhomologous recombination products contained junction-associated sequence insertions. Most insertions resulted from the use of one or more tertiary templates, recognizable as discontiguous portions of viral or host RNA or minus-strand DNA. The donor/acceptor template microidentity evident at most crossovers reconfirmed the remarkable capability of the reverse transcription machinery to recognize short regions of sequence identity. These results demonstrate that recruitment of discontiguous host or viral sequences is a common way for retroviruses to resolve nonhomologous recombination junctions and provide experimental support for the role of splinting templates in the generation of retroviral insertions.