Premium
Specificity determinants for bacteriophage Hong Kong 022 integrase: analysis of mutants with relaxed core‐binding specificities
Author(s) -
Cheng Qiong,
Swalla Brian M.,
Beck Michael,
Alcaraz Renato,
Gumport Richard I.,
Gardner Jeffrey F.
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.01860.x
Subject(s) - integrase , biology , mutant , binding site , bacteriophage , dna , nucleotide , genetics , integrases , site specific recombination , microbiology and biotechnology , recombination , recombinase , escherichia coli , gene
The integrase (Int) proteins encoded by bacteriophages HK022 and λ catalyse similar site‐specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and λ are unable to catalyse recombination between non‐cognate att sites. The att sites of both phages contain weak binding sites for Int, known as ‘core‐type’ sites. Negatively acting nucleotide determinants associated with specific core sites (λ B′, HK022 B′, HK022 C) are responsible for the barrier to non‐cognate recombination. In this study, we used challenge phages to demonstrate that the λ and HK022 Ints cannot bind to core sites containing non‐cognate specificity determinants in vivo . We isolated mutants of the HK022 Int, which bind the λ B′ core site. Two mutants, D99N and D99A, have changed a residue in the core‐binding (CB) domain, which may be directly contacting the core site DNA. We suggest that binding to the λ B′ site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the λ B′ site. We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine λ att sites.