Identification of Cre residues involved in synapsis, isomerization, and catalysis.
The Cre protein of bacteriophage P1 is a tyrosine recombinase and catalyzes recombination via formation of a covalent protein-DNA complex and a Holliday junction intermediate. Several co-crystal structures of Cre bound to its target lox site have provided novel insights into its biochemical activities. We have used these structures to guide the mutagenesis of several Cre residues that contact the lox spacer region and/or are involved in intersubunit protein-protein interactions. None of the mutant proteins had significant defects in DNA binding, DNA bending, or strand-specific initiation of recombination. We have identified novel functions of several amino acids that are involved in three aspects of the Cre reaction. 1) Single mutation of several NH2-terminal basic residues that contact the spacer region of loxP caused the accumulation of Holliday junction (HJ) intermediates but only a modest impairment of recombination. These residues may be involved in the isomerization of the Holliday intermediate. 2) We identified three new residues (Arg-118, Lys-122, and Glu-129) that are involved in synapsis. Cre R118A, K122A, and E129Q were catalytically competent. 3) Mutations E129R, Q133H, and K201A inactivated catalysis by the protein. The function of these Cre residues in recombination is discussed.
Lee
Linda
L
Department of Molecular and Medical Genetics, University of Toronto, Toronto M5S 1A8, Canada.
Sadowski
Paul D
PD
D017101:Bacteriophage P1 / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D013696:Temperature; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases