Effects of Identity Minimization on Moloney Murine Leukemia Virus Template Recognition and Frequent Tertiary Template-Directed Insertions during Nonhomologous Recombination

Abstract

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.Retroviral genetic recombination does not involve nucleic acid breakage and rejoining but instead results from template switching by reverse transcriptase (RT) during viral DNA synthesis (2). Early work demonstrated that the reassortment of retroviral genes is so frequent that even markers approximately 1 kilobase apart behave essentially as if they are unlinked (29). More-recent studies assessing crossover frequencies provide the basis for these observations by demonstrating that recombinogenic template switching likely occurs several times during the synthesis of nearly all retroviral DNAs (21,37,49,72).How elongating reverse transcription complexes recognize and recruit secondary templates and how a growing DNA strand is transferred to an acceptor template are areas of active investigation. Models for recombination during both plus-and minus-strand DNA synthesis have been proposed, with experimental evidence supporting a dominant role for minus-strand recombination (9, 22, 67). Forced copy choice models for minus-strand recombination suggest that RT/primer-template complex dissociation at a broken template end precedes recombinogenic template switching (9). However, evidence that factors other than template integrity can modulate recombination fre...

The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination

“…In the absence of a second gRNA, a broken template or damaged template base would lead to either mutation or aborted synthesis. Findings that the average length of DNA synthesized on single-copy templates appears shorter than average homologous crossover distances are consistent with the possibility that processive RT elongation complexes may include both donor and acceptor templates (87). It is tempting to speculate that some of the cryptic nucleic acid binding sites on exposed surfaces of HIV-1 RT, which complicate efforts to produce uniform preparations of RT poised at the 3Ј ends of primer strands, may contribute to these additional binding functions (137,138).…”

“…One analysis of over 100 nonhomologous crossovers found junctional microhomology in about 90% of crossovers (87). Thus, in contrast to homologous recombination, which is highly efficient and is dependent principally on donor-acceptor identity behind crossover points, nonhomologous recombination is relatively inefficient and dependent largely on primer terminus-mediated acceptor recognition.…”

“…Indeed, RT is capable of significant mismatch extension upon template switching both in vitro and during viral replication (69,80,199,202,254,256,264). At least for MLV, there is a sharp drop-off in the efficiency of acceptor recognition below a threshold homology length that corresponds roughly to the length between RNase H and DNA polymerase active sites and then an additional abrupt drop in recognition for acceptors with only eight or fewer bases of donor template identity (73,87,256). Together, these findings suggest that regions of donor-acceptor homology, behind the growing point for DNA synthesis but contained within the RT footprint, are necessary for efficient homologous recombination.…”

“…Experimentally, many nonhomologous crossovers display a pattern called "insertions in deletions" (87,245,248). Dissection of their structures led to the suggestion that they result from serial nonhomologous recombination and the propensity of the reverse transcription machinery to transfer to regions of microhomology in the absence of more extensive donor/acceptor identity (Fig.…”

“…Nonhomologous recombination, which occurs about 100-fold less (361), often reveals junctional microhomology (87,120,245,248,311,332,360). One analysis of over 100 nonhomologous crossovers found junctional microhomology in about 90% of crossovers (87).…”