Identifying the Nuclease(s) which Degrade Phosphorothioated Cassette Ends.

In order to determine which of the nucleases removed from nuc4− is responsible for cleaving terminal PT bonds from dsDNA, four different strains were created with only three of the four nuc4− nucleases inactivated. VPT1 and VPT4 cassettes were recombined into all strains, and the ratio of number of recombinants calculated. Only the strain containing functional xseA had nearly as many VPT4 recombinants as VPT1 recombinants. This suggests that xseA (ExoVII) is the nuclease responsible for cleaving PT bonds off the end(s) of VPT4, thereby rendering the cassette recombinogenic. EcNR2.xonA−,recJ−,xseA− did not give kanR recombinants for either cassette; this strain was recreated by restoring exoX function in nuc4−, whereupon it exhibited the same non-recombinogenic phenotype. The biological basis of this phenotype is unclear. VPT4:VPT1 ratios for EcNR2, EcNR2.endA−, nuc4−, and EcNR2.xseA− are also shown for comparison.