Specificity determinants for bacteriophage Hong Kong 022 integrase: analysis of mutants with relaxed core-binding specificities.
The integrase (Int) proteins encoded by bacteriophages HK022 and lambda catalyse similar site-specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and lambda 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 (lambda 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 lambda 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 lambda 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 lambda B' site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the lambda B' site. We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine lambda att sites.
Cheng
Q
Q
Department of Microbiology and Biochemistry and College of Medicine, University of Illinois at Urbana-Champaign, IL, 61801, USA.
Swalla
B M
BM
Beck
M
M
Alcaraz
R
R
Gumport
R I
RI
Gardner
J F
JF
D000595:Amino Acid Sequence; D001287:Attachment Sites, Microbiological; D010582:Bacteriophage lambda / Q000201:enzymology / Q000378:metabolism; D004926:Escherichia coli / Q000821:virology; D019426:Integrases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000821:virology*; D017906:Siphoviridae / Q000201:enzymology* / Q000235:genetics; D014758:Viral Core Proteins / Q000378:metabolism*
D014758:Viral Core Proteins; D019426:Integrases