Construction And Catalytic Characteristics Of Hetero-dimer Of T7 ENDO I And P-SSP7 ENDO I

T7 Endonuclease I (T7 Endo I) is a very in-depth studied multi-functional endonuclease, which is coded by Escherichia coli T7 phage gene 3. T7 Endo I is a structure specific nuclease, primarily as a resolvase involved in resolving the Holliday junction of the recombination process. The enzyme also has random nicking activity, cutting off only one chain of the double-stranded DNA randomly; counter nicking activity, cutting off the second chain closed the position of the first nick; and single nucleotide mismatch recognition activity. T7 Endo I is Mg²⁺ dependence, and the Mn²⁺ ions can serves as a substitute, but the enzyme activity in two ions are different. The T7 Endo I family (T7-like Endo I) have a wide range of use: SNP detection; random fragmenting of the genome; isothermal PCR amplification technology and so on. Therefore, depth-study the structure and function of the protein family has a great significance.The T7 Endo I homologous protein P-SSP7 Endo I and Syn5 Endo I from Cyanobacteria phages have been identified and studied. The result shows that the three enzymes have almost the same spatial structure and enzymatic characteristics, but there are still differences. Compared with the sequence of T7 Endo I, the N-terminal and C-terminal peptides of P-SSP7 Endo I and Syn5 Endo I are short, which the C-terminal peptides are very important for the T7 Endo I; and theβ-bridge used for connecting the two activity domains is shorted for three consecutive residues. The three enzymes share the same PD-(E/D)XK motif; and the activity center composed of exactly the same amino acid residues, namely Glu, Asp, Glu and Lys. All the three enzymes' spatial structure is domain-swapping homo-dimer connected byβ-bridge, with two catalytic domains, and the catalytic center is formed by the amino acid residues D55, E65 and K67 of one subunit and E20 of another subunit. The two catalytic domains could synergic catalyze as a resolvase, and work independently as endonuclease.Our purpose of this study is to build the hybrid molecules of T7 Endo I and P-SSP7 Endo I, based on the domain-swapping structures and the characteristics of that the two catalytic domains were formed by two subunits of the protein. The hybrid molecules are constructed by the N-section of one molecule and C-section of another molecule. After con-expression in E. coil, the two hybrid proteins could form a hetero-dimer protein. We analyzed its catalytic characteristic and the difference with T7 Endo I and P-SSP7 Endo I.Based on the structure and catalytic characteristics of the enzyme family, this study designed the PCR primers respectively using the T7 Endo I and P-SSP7 Endo I gene in pET21a-MBP-T7 (pM-T7) and pET21a- MBP-P-SSP7 (pM-P-SSP7) plasmid as a templates. Using fusion PCR, gene TP (ΔPAS) and PT (ΔPAS) was fused by two-step PCR respectively. The recombinant plasmid pET28a-His-TP(ΔPAS) (pHTP(ΔPAS)) and pET21a-MBP-PT(ΔPAS) (pMPT(ΔPAS)) was constructed. Expressed the two recombinant plasmids in E2566 together and then the dual-label hetero-dimer His-TP(ΔPAS) / MBP-PT(ΔPAS) (TP*) was purified by Amylose and Ni-NTA affinity chromatography. The activities of the hetero-dimer were analyzed in Mg²⁺ and Mn²⁺ buffer respectively, including: resolving activity, random nicking activity, counter nicking activity and single nucleotide mismatch recognizing activity.Our study first demonstrated the technology of construction a hetero-dimer of T7 Endo I and P-SSP7 Endo I. The results showed that the hetero-dimer was basically full activity, and showing different activity in Mg²⁺ and Mn²⁺ respectively. In Mg²⁺, the enzyme showed mainly resolving and counter nicking activity, about 4 and 8 times higher than in the Mn²⁺. In Mn²⁺, the enzyme showed mainly random nicking, about 8 times higher than in the Mg²⁺; and a good single nucleotide mismatch recognizing activity, almost recognizing all different mismatch. Our experiments found that the hetero-dimer had its own activity and metal ion dependence characteristics, it's not the neutralization or superposition of the characteristics of T7 Endo I and P-SSP7 Endo I. The experimental results of this study provided a idea for future to construct more specific functional hetero-dimeric nucleases. And our result provided a new way to enhance the concentration of cytotoxicity protein T7-like Endo I using the method of that expressing the two hybrid proteins apartly and denaturating/ renaturating together to form a hetero-dimer.