UBC Theses and Dissertations
Essential roles of the T7 Endonuclease (Gene 3) and the T7 Exonuclease (Gene 6) in recombination of Bacteriophage DNA Lee, Marion A.
The role of the T7-induced exonuclease (gene 6) in recombination was studied using both molecular and genetic techniques. In the molecular method the fate of parental DNA during parent-to-progeny recombination was examined. A comparison of infections with T7⁺, T7am6 (amber gene 6), or T7ts6 (temperature sensitive gene 6) under permissive and nonpermissive conditions was made. CsCl density gradient analysis of replicative DNA indicated that the T7 exonuclease is necessary for recombination to occur, i.e., in the absence of the exonuclease the parental DNA replicated continuously as a hybrid molecule and did not recombine. Analysis of denatured replicative DNA by CsCl density gradient centrifugation indicated that the exonuclease also may be needed for a limited amount of covalent repair of recombinants. Further confirmation of the essential role which the exonuclease plays in recombination came from genetic analysis. The T7 exonuclease was shown to be necessary for intragenic and intergenic recombination in several areas of the T7 genetic map; genetic recombination frequencies were found to be decreased from 3 to 18-fold under conditions nonpermissive for the exonuclease. The role of the T7-induced endonuclease (gene 3) in molecular recombination was studied by examining the fate of parental DNA during parent-to-progeny recombination using a shear technique. The T7 endonuclease was found to be necessary for the dispersion of parental DNA in the newly replicated DNA. Concatemers synthesized by either T7⁺ or T7am3 (amber gene 3) phage containing the newly replicated DNA were sheared to the size of mature phage DNA and also to quarter size molecules. In the presence of gene 3 protein, parental DNA and newly replicated DNA were interspersed, i.e., the 32P-label from the sheared DNA was found to sediment at the density of recombined DNA. In the absence of gene 3 protein, the parental strand of each sheared DNA molecule was usually found intact, i.e., the ³²P-label from the sheared DNA was found to sediment at the density of hybrid DNA. These results support the previous genetic data (52, 83) that the gene 3 protein is essential for T7 recombination. The role of T7 recombination enzymes in the formation of concatemers was studied by examining selected gene 3 and gene 6 mutants. Results of sucrose gradient analysis showed that DNA concatemers were formed when both the T7 exonuclease (gene 6) and the T7 endonuclease (gene 3) were absent. Further results showed that concatemers cannot be maintained in the absence of the exonuclease unless the endonuclease was eliminated. In a T7am6 infection DNA concatemers formed early were prematurely broken down and accumulated as fragments smaller than mature size phage DNA. In a T7am3am6 (amber in both genes 3 and 6) infection concatemers accumulated and were not matured. These results indicate that concatemers are formed by a process other than normal phage recombination. However, selective defects in the recombination system do interfere with the stability of concatemers.