Gamma integrase complementation at the level of DNA binding and complex formation.
Site-specific recombinases of the gamma Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites. Each protomer may cleave its own site as a monomer in cis (as for Cre recombinase), or it may recruit the tyrosine from its partner in trans to form a composite active site (as for Flp recombinase). The crystal structure of the gamma Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on gamma integrase (Int) generated data that were not in agreement. Support for cis and trans cleavage came from assays with bispecific DNA substrates for gamma and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence for cis cleavage, but they strongly suggest that the previously described complementation results cannot be used in support of a trans-cleavage mechanism. We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-order attL intasome structures. The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions. Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present. This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis. Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (in cis) by mutant gamma Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the gamma Int mutants.
Nunes-Düby
Simone E
SE
Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Radman-Livaja
Marta
M
Kuimelis
Robert G
RG
Pearline
Rachel V
RV
McLaughlin
Larry W
LW
Landy
Arthur
A
D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism*; D005816:Genetic Complementation Test; D019426:Integrases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic
D004279:DNA, Viral; D009841:Oligonucleotides; D019426:Integrases