Mutational analysis of highly conserved residues in the phage phiC31 integrase reveals key amino acids necessary for the DNA recombination.
BACKGROUND
Amino 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 recombination of phiC31 integrase.


RESULTS
To 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
This 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.
Liu
Shaohui
S
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China.
Ma
Jinfang
J
Wang
Wei
W
Zhang
Maoxiang
M
Xin
Qingting
Q
Peng
Siman
S
Li
Rongxiu
R
Zhu
Huanzhang
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001435:Bacteriophages / Q000201:enzymology*; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics*; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D017931:DNA Primers; D004279:DNA, Viral; D019426:Integrases