Abstract
BackgroundAmino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase.Methodology/Principal FindingsTo determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage.Conclusions/SignificanceThis data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.
Highlights
The integrase from the bacteriophage phiC31 was first described in 1991 as a 613 amino acid open reading frame recombinase [1]
We observed that mutants G182A/F183A, C374A, C376A/G377A, Y393A and V566A had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein, as no complexes were seen in the gel retardation experiment (Fig. 5A)
Sequence of phiC31 integrase can be aligned with other members of the serine recombinase family and it contains approximately 62 highly conserved amino acids residues
Summary
The integrase from the bacteriophage phiC31 was first described in 1991 as a 613 amino acid open reading frame recombinase [1]. The phiC31 integrase recognizes native sequences in human and mouse genomes that possess partial sequence identity to attP, called pseudo attP sites, and mediates the integration of plasmids bearing an attB site into such pseudo attP sequences [6,7]. This ability of phiC31 integrase to integrate into endogenous genomic sites has been used in gene therapy applications[8,9,10,11] and engineering human embryonic stem cell lines and primordial germ cells [12,13]. No system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase
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