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. 1997 Jun;179(12):4030–4038. doi: 10.1128/jb.179.12.4030-4038.1997

Genetic analysis of second-site revertants of bacteriophage lambda integrase mutants.

Z Wu 1, R I Gumport 1, J F Gardner 1
PMCID: PMC179214  PMID: 9190821

Abstract

Bacteriophage lambda site-specific recombination is catalyzed by the phage-encoded integrase (Int) protein. Using a collection of 21 recombination-defective Int mutants, we performed a second-site reversion analysis. One of the primary mutants contained a valine-to-glutamic acid change at position 175 (V175E), and a pseudorevertant with a lysine change at this site (V175K) was also isolated. Relative to the wild-type 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 containing an alanine substitution (V175A) was made by site-directed mutagenesis, 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 second primary mutant contained a proline-to-leucine change at position 243 (P243L). A true second-site revertant was isolated that contained a glutamic acid-to-lysine change (E218K). The P243L-E218K protein promoted recombination and bound arm-type sites more efficiently than the original P243L protein but not as efficiently as the protein containing the E218K substitution alone. The E218K substitution also restored activity to a mutant with a threonine-to-isoleucine substitution at position 270 (T270I). This result showed that suppression by the E218K change is not allele specific and suggests that the substitution improves an inherent activity of Int rather than directly compensating for the defect caused by the primary substitutions. Results with challenge phages carrying attL sites with altered core sites indicate that the E218K change may improve binding to the core site.

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