mutational analysis of highly conserved residues in the phage phic31 integrase reveals key amino acids necessary for the dna recombination突变分析的高度保守的残留噬菌体phic31整合酶显示关键的dna重组所必需的氨基酸.pdfVIP

Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination 1 1 1 1 1 1 2 Shaohui Liu , Jinfang Ma , Wei Wang , Maoxiang Zhang , Qingting Xin , Siman Peng , Rongxiu Li , Huanzhang Zhu1* 1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China, 2 MOE Key Laboratory of Microbial Metabolism, and School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China Abstract 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 recombinaton of phiC31 integrase. Methodology/Principal Findings: 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.