Large - Scale Directed Mutagenesis Of Conserved Residues Of φC31 Site - Specific Integrase And Identification And Functional Analysis Of Its Zinc Finger Domain

Streptomyces phage OC31integrase was reported to be able to mediate exogenous DNA containing attB sequences site-specific integration into pseu-attP sites on mammalian cellular genome in the case does not require any cofactors. It is possible that the φC31integrase system has great potential for a tool of gene manipulation and gene therapy. However, the specificity and efficiency of φC31integrase integration in mammals need to be further improved. Analysis the relationship between structure and function of the φC31integrase is the basis and key for solving above problems. In this paper, we studied the relationship between the structure and function of the φC31integrase by bioinformatics prediction at the massive conservative sites mutation and the zinc finger domain analysis two aspects.In the first part of present study, amino acid sequence alignment of phage phiC31integrase with the serine recombinases family revealed62highly conserved residues outside the catalytic domain. To determine the functional roles of these conserved residues、19conserved residues were targeted by site-directed mutagenesis, and17mutants were obtained. Out of the17mutants,11mutants (R18A、I141A、 L143A、 E153A、 G182A/F183A、 C374A、 C376A/G377A、 Y393A、 I432A、 V566Aand V571A) showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. To analyze the cause of decreased integrase activity of the11mutants, the substrate binding activity was measured by electrophoretic mobility shift assay (EMSA). Results of DNA binding activity assays showed that6mutants (R18A, I141A, L143A, E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and5mutants (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. To investigate whether the mutants R18A,1141A, L143A, E153A and I432Athat show the lower affinity for the att sites also affect the synapsis and cleavage steps, DNA synaptic complexes and cleavage ability of the proteins were measured by EMSA.The result showed that these mutants were blocked in recombination at the stage of strand cleavage. To determine if the secondary structure of these proteins had been significantly affected by the point mutations, We compared the circular dichroism (CD) spectra of the wild-type and mutant proteins losing DNA binding activities.The result showed that mutants C374A and C376A/G377A did not result in significant changes in the protein secondary structure, however, mutants G182A/F183A, Y393A, V566A and V571A had a significant change at β-sheets and turn, respectively, indicating that mutations of these residues were likely to affect proper folding of the β-strands into a hairpin structure. Collectively, these data reveals for the first time that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the leu/val-rich region in the C-terminal region of phiC31integrase may represent the major DNA binding domain of phiC31integrase.In the second part of present study, amino acid sequence alignment of phage phiC31integrase with the serine recombinases family and other zinc finger proteins revealed a zinc finger domain exist in ΦC31integrase, and the zinc atom was combined by the four cysteines (C374, C376, C395and C413). In order to verify the presence of OC31integrase zinc finger domain, we obtained four mutations (C374A, C376A, C395A and C413A)at the four conserved cysteines to alanines by site-directed mutagenesis. We have applied ICP-MS and the results showed1mole wild type ΦC31integrase containing0.99mole of zinc atoms.In contrast, the content of zinc ion was significantly reduced in the C374A, C376A,C395A and C413A mutants containing<0.4mol of zinc/mol protein. This result confirms the above assumptions that ΦC31integrase enzyme exists a zinc finger domain witch consolidated by the C374, C376, C395, and C413four cysteines binding with one zinc atom. In order to determine the importance of the zinc finger domain inΦC31integrase conformational stability, we performed limited proteolysis on both wild-type ΦC31integrase and cysteine mutants. The result shows that cysteine mutants C374A, C376A, C339A are less resistant to proteases than the wild-typeΦC31integrase, and suggests the protein conformation stability of mutants C374A, C376A, C395A is lower than the wild-type. To determine the functional roles of zinc finger domain in ΦC31integrase, recombination ability of cysteine mutants were analyzed by recombination assay in vivo. The results showed that mutants C374A, C376A, C395A showed no recombination ability, and the recombination ability of mutants C413A is40.4%. The results suggest that damage of zinc finger domain affecting to φC31integrase recombination activity.To confirm the recombinant activity reduction of cysteine mutants was caused by DNA binding ability reduction, We tested the DNA binding affinity of these mutants to both attB and attP. The results showed that not all these Cysteins were equally important to DNA binding. C374was seen vital for both attB and attP binding. C376was seen less important than C374, however, more than C395and C413in attP binding. C376and C395were also vital for attB binding and C413contributed the least to both attB and attP binding indicated by the less affected DNA binding affinity. This indicates that the φC31integrase zinc finger domains play a role in DNA recognition and binding. Mutations(φC31(△383-399). R384A, K390A、R394A、 R396A、R397A、 R398A and R399A) at basic amino acids in zinc finger domain to alanines decrease its DNA binding activity and recombination activity in vivo.This indicates OC31integrase may identify the specific sequence of the attB and attP by basic amino acid in zinc finger domain. AttP-Emut can not able to bind with φC31integrase.This indicates that the E-box-like sequence in attP play an important role in the binding with φC31integrase.In summary, we studied the massive conservative sites mutation and the zinc finger domain analysis two aspects thlough the bioinformaties structure prediction and biologieal techniques. Our results show that mutation of some highly conserved residues lead to the loss of recombination activity and that these defects are due to impaired DNA binding affinity or affect specific target DNA cleavage, indicating that these conserved residues both in the N-terminus and C-terminus region of phiC31integrase are essential in DNA recombination. We also confirmed the existence of zinc finger domain in φC31integrase, and the zinc finger domain plays the main role of DNA binding in φC31integrase. This finding lays the foundation for further understand the structure and function of φC31integrase, and in particular developed a higher specificity and efficiency of the φC31integrase system widely used in gene therapy.