EFFECTS OF DNA SUBSTRATES ON RECOMBINATION IN E. COLI

The level of recombination seen in E. coli with F421ac times Lambdaplac5 transduction is fiftyfold higher than is observed with Lambdaplac5 transduction of a chromosomal lac gene. The enhanced recombination with F421ac is a cis-dominant, tra-dependent phenomenon. Mini-F-lac plasmids will be constructed to determine the location of the site on F421ac where the tra function(s) act to facilitate the initiation of recombination. Other tra containing plasmids will be used for in-trans complementation on mini-F-lac derivatives in vivo in order to determine which tra gene product(s) are involved in this mechanism. Comparisons of recombination initiation with conjugation and transduction in E. coli have indicated that the single-strandedness of DNA transferred by conjugation bypasses the need for the recBC gene product in recombination initiation. The kinetics of recombination in heterozygous lacZ merodiploids that are converted from recA- to Rec+ by infection with LambdaprecA+ will be studied in recB- and recB+ strains to further examine this question. The fundamental comparison will be between stable heterozygous lacZ merodiploids and merodiploids just formed by a conjugational transfer of F421ac. The mechanistic differences between the three putative recombination pathways in E. coli are poorly understood. Specialized transduction studies with genetically disabled Lambdaplac5 derivatives allows the nature of the recombination end-product to be determined by assaying addition and substitution transductants. Such studies will be utilized to compare the mechanisms employed by the RecBC, RecE and RecF recombination pathways in E. coli. These studies should yield significant information regarding the initiation and the mechanism of genetic recombination which is an important but poorly understood aspect of DNA metabolism.