Study On Phage Resolvase T7EI,P-SSP7EI & Syn5EI

Genetic recombination is one of the most important driving forces of evolution. Organisms can insert, delete and rearrange genes and traits, repire DNA lesion and can maintain the dynamic stability of genetic information. Resolving of the intermediate Holliday Junction（HJ）is the key step during recombination, and the enzyme involved in this process was known as resolvase. Resolvases are endonucleases, which are widely distributed in all kinds of life-form, such as viruses, prokaryotes and eukaryotes, and known resolvases exist as homodimers. They can specific recognize and resolve the four-way DNA junction to two nicked duplex DNA by simultaneously introducing two nicks on two strands. T7 endonucleases I（T7EI）was encoded by gene 3 of T7 phage and it is one of most studied resolvases. Now, it is not only a model enzyme for research about resolving mechanism, but also a model molecular for research about amyloid protein deposition.Cyanobacteria are the origin of chloroplasts, they have the capability of photosynthesis and biological nitrogen fixation, they are the producer of the original product and they are widely distributed in the surface oceans. In recent years, research about cyanobacteria and cyanophage genomics has provide knowledge about co-evolution between them. Cause the origin of species in ocean, the genomes of cyanobacteria and cyanophage provide new materials for evolution study and a new resource for new enzymes, which can be used as tools for genetic engineering.This study focused on T7EI and its homologes from cyanophages P-SSP7 endonuclease I（P-SSP7EI）and Syn5 endonuclease （ISyn5EI）. There are two topic of research were carried on carefully. One is the dimerization of T7EI, another is the catalytic properties of cyanophage endonuclease I. The details were listed below.1. In order to analyze the binding strength and dimerization pattern of T7EI, a series of heterodimer T7EIs with one active domain were designed, expressed and purified, and a affinity chromatography based approach was setup to detect the binding strength of the different dimers. Results show that the heterodimers have nick endonuclease activity, and they can bind with Junction3 specifically. Competitive refolding results that the higher binding strength the easier to refold efficiently, and mutations at theβ-bridge can significant impact on the stability and refolding efficiency of T7EIs heterodimer. Based on these results, we proposed a hypothesis. During dimerization, theβ-bridge dimerized firstly to form a folding intermediate, then two active domain fold sequentially. Using this hypothesis upon results can be explained, and we can give a reasonable explanation for the question:“Why the T7EI exists as a dimer under physiological conditions and does not fold into amyloid protein deposition?”2. Gene 3 of cyanophage P-SSP7 encodes a 116 amino acids homologous of T7EI（149aa）, P-SSP7 endonuclease I（P-SSP7EI）. Compared to T7EI, P-SSP7EI does not contain the N-terminal 13 amino acids and the C-terminal 18 amino acids are missing. Theβ-bridge sequence of P-SSP7EI is shorter by 3 amino acids. In T7EI, deletion of C-tail and modification atβ-bridge can lead to enzyme inactivation and activity changing. In this study, Gene3 of P-SSP7 was assembled by PCR and cloned into pET21a and pET28a vectors. MBP and His-tag fusion expression systems were used and tag-removed pure enzyme was obtained finally. Results show:（1）P-SSP7EI can specific recognize and cleavage cruciform substrate pUC（AT）and four-way junction substrate Junction3. P-SSP7EI′s binding capacity to Junction3 is about 1000 times to duplex DNA, and the dissociate constant（kd）of enzyme-Junction3 complex is 10nM.（2）P-SSP7EI has Random-Nick activity on one strand of duplex DNA in Mg²⁺ buffer. P-SSP7EI not only has Random-Nick activity but also Count-Nick activity in Mn²⁺ buffer. The counter-nick cleavage site is around 3～4 nucleotide of the 5′stream（.3）In Mn²⁺ buffer, P-SSP7EI can cleave single nucleotide mismatch, and it can cleave all type of mismatch. But in Mg²⁺ buffer, only 4 type can be cleaved.（4）Site mutation confirm that Glu7、Asp39、Glu49、Lys52 form the active site of P-SSP7EI. Inserting 3 amino acids at theβ-bridge leads to inactivation,and adding extra C-tail to P-SSP7EI can not change the enzyme activity dramatically.（5）Mn²⁺ can significantly improve the themo-stability of P-SSP7EI.3. Gene 22 of cyanophage Syn5 encodes a 118 amino acids homologous of T7EI. Protein sequence comparison shows a 46% amino acid identity between Syn5EI and P-SSP7EI, and 44% between Syn5EI and T7EI. Compared to T7EI, Syn5EI does not contain the N-terminal 11 amino acids and the C-terminal 17 amino acids are missing. Theβ-bridge sequence of Syn5EI is shorter by 3 amino acids. In this study, gene22 of Syn5 was assembled and cloned. MBP and His-tag fusion expression systems were used and tag-removed pure enzyme was obtained finally. Results show: Similar to P-SSP7EI and T7EI, Syn5EI can specific recognize and cleave Holliday junction structure. Syn5EI has Random-Nick activity on duplex DNA in Mg²⁺ buffer. Syn5EI not only has Random-Nick activity but also Count-Nick activity in Mn²⁺ buffer.4. Phylogenetic tree of phage endonucleaseⅠprotein family was constructed, and protein structure evolution of this protein family was analyzed. Results suggest that there is a special relationship between phage endonucleaseⅠand their hosts′living environment. A remote homologe of T7EI, phiv10-p45 was cloned and expressed. It has not resolving activity but only nicking activity. Phiv10-p45 belongs to a uncharacterized protein family Pfam06356 with PD……（E/D）XK motif. This study provides function annotation for them for the first time.In this study, heterodimer T7EIs with one active domain was designed and obtained, the binding strength and refolding efficiency of heterodimers were characterized, and a hypothesis about T7EI dimerization was proposed. This study provide some new knowledge about molecular design, protein dimerization and amyloid protein deposition. In this study, cyanophage resolvase P-SSP7EI and Syn5EI were cloned, expressed and purified first time, and their catalytic properties were characterized comprehensively. This study extends the scope of phage resolvase, provide new detail about resolving mechanism. These enzymes can be used as potential tools for research and biotechnology industry.