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

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 recombination 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.

Conclusions/significance: 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.

Figure 1. Sequence alignment of eight members including phiC31 integrase within large serine integrase family.

The conserved residuals are shown in colour. Positions mutated in this study are indicated by asterisks. Sequence alignment was accomplished by using VNTI 9.0.

Figure 2. Recombination assay of phiC31 mutants in E. coli.

Each mutant plasmid and its report plasmid were co-transformed into Top10 bacteria, respectively. The bacteria were then plated to form colonies on double antibiotic selecting agar plates containing X-Gal. When mutant recombinases still have their recombination ability, this recombination event consequently produces white colonies (A). When mutant integrase have lost or reduced recombination ability, It is observed that mixture of white and blue colonies (B) or only blue coloneies (C) on the plates. To further verify site-specific recombination at molecular level, the white colonies were picked, and plasmid DNA was subjected to PCR with specific primers that would amplify a 400-bp product only from the recombinant. DNA from 5 white colonies was subjected to PCR with specific primers, and a 400-bp product got amplified (D). The sequence of this PCR product contained the predicted chimeric attL site, comprising an attP arm (left) and attB arm (right) around the core AA dinucleotides (gray ellipse).

Figure 3. Recombination assay of the purified wild-type and mutant phiC31 integrases in vitro.

(A) Schematic representation of functional phiC31 integrase assay. The linear 4.9 kb DNA substrate (PB-L) containing correct attB and attP sites was treated with wild-type or mutants phiC31 integrase, resulting in irreversibly produce a circular pBCSK molecule (or pBC-attL) and a 1.4 kb linear attR fragment (attR-L). (B) A 0.04 µM linear DNA substrate (PB-L) was treated with a 0.1 µM purified wild-type or mutant phiC31 integrase in recombination buffer at 30°C for 30 min and then stopped at 75°C for 10 min. The recombination reaction products were separated by electrophoresis on a 1% agarose gel. Arrows show the positions of circular molecule (solid line arrows), and a linear attR fragment (dotted line arrows). Wt is wild-type.

Figure 4. Assay of DNA binding activity of wild-type phi C31 integrase.

A fixed amount (16 fmol) of a 41 bp biotin -labeled attB fragment (A) or a 50 bp biotin -labeled attP fragment(B) was incubated with varying amounts of purified wild -type phi C31 in binding buffer at 30°C for 30 min. Reactions were analyzed by polyacrylamide gel electrophoresis (5% polyacrylamide, 1x Tris-borate-EDTA). Arrows show the positions of free probe (F) and the DNA binding complexes (I, II and III).

Figure 5. Assay of DNA binding activity of mutant phi C31 integrases.

A fixed amount (16 fmol) of a 41 bp biotin -labeled attB fragment (A) or a 50 bp biotin -labeled attP fragment (B) was incubated with a similar amount of purified wild -type and mutant phi C31 in binding buffer at 30°C for 30 min. Results were analyzed by polyacrylamide gel electrophoresis (5% polyacrylamide, 1x Tris-borate-EDTA). Arrows show the positions of free probe (F) and the DNA binding complexes (I, II and III).

Figure 6. DNA synapsis and cleavage assays with wild-type and mutant integrases.

Labelled attB was incubated with wild-type or mutant integrases (Panel A:R18A, I141A; Panel B:L143A, E153A; Panel C:I432A) in the presence of no cold attP, 198 bp cold attP in binding buffer and then untreated or treated with proteinase K. Reactions were analyzed by polyacrylamide gel electrophoresis (5% polyacrylamide, 1x Tris-borate-EDTA). The complexes observed are integrases (Int) bound to attB or synaptic complexes (SC). The recombinant products attL and attR are also indicated.

Figure 7. FT-IR spectra of the phiC31 integrase in heavy water.

The second derivative of the infrared spectrum (A) and the absorption profile of the amide I' band (B).

Figure 8. CD spectra of wild-type and mutant phiC31 proteins.

Far UV spectra from 190 to 250 nm of wild-type phiC31 and mutants G182A/F183A, C374A, C376A/G377A, Y393A, V566A and V571A were shown, at 25°C using a 0.02-cm quartz cell. Protein was dissolved in 0.15 M sodium chloride, pH 7; Other conditions were described in Experimental Procedures. Wt is wild-type.