Effects of DNA structure and homology length on vaccinia virus recombination

Replicating poxviruses catalyze high-frequency recombination reactions by a process that is not well understood. Using transfected DNA substrates we s how that these viruses probably use a single-strand annealing recombination mechanism. Plasmids carrying overlapping portions of a luciferase gene exp ression cassette and luciferase assays were first shown to provide an accur ate method of assaying recombinant frequencies. We then transfected pairs o f DNAs into virus-infected cells and monitored the efficiencies of linear-b y-linear, linear-by-circle, and circle-by-circle recombination. These exper iments showed that vaccinia virus recombination systems preferentially cata lyze linear-by-linear reactions much more efficiently than circle-by-circle reactions and catalyze circle-by-circle reactions more efficiently than li near-by-circle reactions. Reactions involving linear substrates required su rprisingly little sequence identity, with only 16-bp overlaps still permitt ing similar to4% recombinant production. Masking the homologies by adding u nrelated DNA sequences to the ends of Linear substrates inhibited recombina tion in a manner dependent upon the number of added sequences. Circular mol ecules were also recombined by replicating viruses but at frequencies 15- t o 50-fold lower than are linear substrates. These results are consistent wi th mechanisms in which exonuclease or helicase processing of DNA ends permi ts the forming of recombinants through annealing of complementary single st rands. Our data are not consistent with a model involving strand invasion r eactions, because such reactions should favor mixtures of linear and circul ar substrates. We also noted that many of the reaction features seen in viv o were reproduced in a simple in vitro reaction requiring only purified vac cinia virus DNA polymerase, single-strand DNA binding protein, and pairs of linear substrates. The 3'-to-5' exonuclease activity of poxviral DNA polym erases potentially catalyzes recombination in vivo.