GENETIC-ANALYSIS OF 2ND-SITE REVERTANTS OF BACTERIOPHAGE-LAMBDA INTEGRASE MUTANTS

Bacteriophage lambda site-specific recombination is catalyzed by the p hage-encoded integrase (Int) protein. Using a collection of 21 recombi nation-defective Int mutants, we performed a second-site reversion ana lysis. One of the primary mutants contained a valine-to-glutamic acid change at position 175 (V175E), and a pseudorevertants with a lysine c hange at this site (V175K) was also isolated. Relative to the wild-typ e protein, the V175E protein was defective in its ability to form the attL complex and to catalyze excision in vivo and in vitro. A mutant c ontaining an alanine substitution (V175A) was made by site-directed mu tagensis, and it was more efficient than the V175K protein in forming the attL complex and promoting excision. These results indicate that a nonpolar side chain at residue 175 is required for function. The seco nd primary mutant contained a proline-to-leucine change at position 24 3 (P243L). A true second-site revertant was isolated that contained a glutamic acid-to-lysine change (E218K). The P243L-E218K protein promot ed recombination and bound arm-type sites more efficiently than the or iginal P243L protein but not as efficiently as the protein containing the E218K substitution alone. The E218K substitution also restored act ivity to a mutant with a threonine-to-isoleucine substitution at posit ion 270 (T270I). This resulted showed that suppression by the E218K ch ange is not allele specific and suggests that the substitution improve s an inherent activity of Int rather than directly compensating for th e the defect caused by the primary substitutions. Results with challen ge phages carrying attL sites with altered core sites indicate that th e E218K change may improve binding to the core site.