CN114807086B

CN114807086B - Holliday connector dissociating enzyme DrRuvC and encoding gene and application thereof

Info

Publication number: CN114807086B
Application number: CN202210399536.XA
Authority: CN
Inventors: 程凯莹; 杨婕妤; 孙亦阳
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2022-04-15
Filing date: 2022-04-15
Publication date: 2024-02-20
Anticipated expiration: 2042-04-15
Also published as: CN114807086A

Abstract

The invention discloses a holliday connector dissociating enzyme DrRuvC and a coding gene and application thereof. DrRuvC is derived from deinococcus radiodurans, is in a homodimeric state in solution, can specifically bind to the DNA of the holliday connector, and symmetrically causes single-chain phosphodiester bonds to break at the joint, so that one holliday connector is dissociated into two double-chain DNA with gaps. The holliday linker specifically recognized by the holliday linker contains a conserved sequence of 5'-TC-3' in the connecting junction region, and the enzyme cleavage site is a phosphodiester bond after cytosine. The enzyme prefers 5'- (G/C) TC (G/C) -3' sequence, and the optimal catalytic metal ion is Mn ²⁺ (2.5-10 mM), the optimal reaction pH is 7.6-8.4, the optimal reaction NaCl concentration is 50-150mM, and the optimal reaction temperature is 40-45 ℃.

Description

Holliday connector dissociating enzyme DrRuvC and encoding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, relates to a holliday connector dissociating enzyme DrRuvC, and a coding gene and application thereof, in particular to preparation of a novel holliday connector dissociating enzyme (DrRuvC) from deinococcus radiodurans, and relates to a holliday connector DNA conserved sequence characteristic identified by the enzyme and a method for improving enzyme activity.

Background

The holliday linker (Holliday junction resolvase) appears in the late stage of cellular DNA homologous recombination (homologous recombination) and during the back-off of the DNA replication fork (DNA replication fork regression), and its main structure is a four-chain DNA intermediate rich in homologous regions. Holliday linkers in the genome, if not dissociated efficiently, will cause a disturbance in genome replication, and thus, one or more holliday linker dissociases are often encoded in the cell to help maintain stability of the genome and order of cell division.

Researchers have discovered, several years ago, that the protein encoded by the ruvC gene, ecRuvC, in escherichia coli possesses holliday linker dissociating enzyme activity (Saito, a.et al (1995), "Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.,".Proc Natl Acad Sci U S A92 (16):7470-7474). Ec ruvc is a homodimeric structure that favors the recognition of holliday linkers containing the conserved sequence 5'- (a/T) TT (G/C) -3' in the junction region. The ecluvc can cleave the phosphodiester linkage of the single-stranded region (specifically, the phosphodiester linkage after the second thymine) symmetrically at the holliday linker junction, thereby dissociating one holliday linker into two nicked double-stranded DNA.

Most of the currently commercialized holliday linker dissociating enzymes are recombinant e.coli ec ruvc proteins expressed in vitro, such as the recombinant ec ruvc protein sold by abcam corporation (cat No. P0a 814). The eclovc has a limitation in use as a tool enzyme in genetic engineering because it preferentially dissociates hollydi linker containing 5'- (a/T) TT (G/C) -3' and has a weak ability to dissociate hollydi linker containing other sequences.

Disclosure of Invention

The invention aims to provide a novel holliday connector dissociating enzyme (DrRuvC) derived from deinococcus radiodurans, and a coding gene and application thereof.

The above object of the present invention is achieved by the following technical solutions: amplifying a gene fragment encoding DrRuvC from a genome of the deinococcus radiodurans, constructing a DrRuvC expression vector, preparing a DrRuvC protein, verifying the activity of the DrRuvC protease, screening the optimal substrate sequence of the DrRuvC protein, and searching the optimal enzyme activity conditions (optimal metal ion and concentration, optimal pH, optimal NaCl concentration and optimal temperature) of the DrRuvC.

In a first aspect, the present invention provides a hollidar linker dissociating enzyme DrRuvC, the amino acid sequence of which is shown in SEQ No.4.

In a second aspect, the present invention provides a gene encoding the holliday junction dissociating enzyme DrRuvC, the nucleotide sequence of which is shown in SEQ No.3.

In a third aspect, the invention provides an expression vector comprising a gene encoding DrRuvC.

In a fourth aspect, the invention provides the use of the enzyme DrRuvC to a holliday linker comprising a 5'- (G/C) TC (G/C) -3' sequence at the dissociative junction.

In a fifth aspect, the present invention provides a method for the dissociation of the holliday linker, in particular by placing 10 μl of the reaction system at 40-45 ℃ for 30 minutes, followed by two methods for termination and identification of the product as desired: adding an equal volume of stop solution A to stop the reaction, heating at 100 ℃ for 20 minutes, transferring to ice for quenching, and then separating the product by utilizing TBE urea gum; or, adding an equal volume of stop solution B to stop the reaction, and separating the product by using TBE active gel after stopping the reaction at 37 ℃ for 20 minutes.

The 10 μl reaction system contains 1 μM of the enzyme DrRuvC of claim 1, holliday linker substrate containing 5'- (G/C) TC (G/C) -3' sequence at 500nM junction, 50-150mM NaCl, a concentration of metal ion, 20mM Tris pH7.6-8.4, 0.5mM TCEP, 5% glycerol;

the metal ion is Mn or Mg.

Preferably, the stop solution A is 20mM EDTA and 98% formamide.

Preferably, the stop solution B is 20mM EDTA and 10mg/mL proteinase K.

Preferably, when the metal ion is Mn, the concentration of Mn ion in the reaction system is 2.5-10mM.

Preferably, when the metal ion is Mg, the concentration of Mg ion in the reaction system is 20mM.

Preferably, the pH of the reaction system is 8.0.

The beneficial effects of the invention are as follows:

the novel holliday connector dissociating enzyme DrRuvC is derived from deinococcus radiodurans, is in a homodimeric state in a solution, can specifically bind to the DNA of the holliday connector, and symmetrically causes single-chain phosphodiester bond rupture at the joint, so that one holliday connector is dissociated into two double-chain DNAs with gaps. The holliday linker specifically recognized by the holliday linker contains a conserved sequence of 5'-TC-3' in the connecting junction region, and the enzyme cleavage site is a phosphodiester bond after cytosine. The enzyme prefers 5'- (G/C) TC (G/C) -3' sequence, and the optimal catalytic metal ion is Mn ²⁺ (2.5-10 mM), the optimal reaction pH is 7.6-8.4, the optimal reaction NaCl concentration is 50-150mM, and the optimal reaction temperature is 40-45 ℃.

The novel holliday connector dissociation enzyme DrRuvC disclosed by the invention has the advantages that the preferential substrate sequence is different from that of the existing commercialized holliday connector dissociation enzyme (EcRuvC), the substrate limitation problem of the holliday connector dissociation enzyme is solved, and the novel holliday connector dissociation enzyme has important significance in developing novel molecular biology tool enzymes.

Drawings

FIG. 1 shows the detection of drruvC gene fragments amplified from the genome of P.radiodurans by agarose gel. The amplified target fragment was about 550bp in size.

FIG. 2 is a map of the pET28T-drruvC expression vector after successful construction.

FIG. 3 (A) shows a map of the purified DrRuvC molecular sieve (Superdex 75) with peak positions at 10ml, corresponding molecular weights of about 40-45kDa, and the molecular weight of the DrRuvC monomer at about 20kDa, demonstrating that DrRuvC exists as a homodimer in solution; (B) purity of DrRuvC was checked by 15% SDS-PAGE.

Fig. 4 is a test of the effect of DrRuvC on holliday connector dissociation. Above the picture is the synthetic holliday linker substrate HJ98 used in this experiment, the portion of the substrate sequence that is plotted as a horizontal line being the region of homologous sequence of the holliday linker. The sequence without a frame on the gray background is a specific enzyme digestion sequence of the commercialized EcRuvC enzyme, and the sequence with a frame on the gray background is a specific enzyme digestion sequence of the DrRuvC enzyme prepared in the invention. Because the 5' end of the corresponding sequence in the substrate is provided with a 6-FAM fluorescent group, the enzyme digestion product of the sequence can be detected by a fluorescent glue scanner with corresponding wavelength after being separated by 15% TBE urea glue. Among them, ec ruvc is a recombinant ec ruvc protein sold by abcam corporation as a control. The leftmost lane of the gel is a single-stranded DNAmarker.

Fig. 5 is an analysis of holliday linker dissociation sites by DrRuvC. Based on the results of FIG. 4, a shorter holliday linker substrate (HJ 31) was designed to more precisely locate the DrRuvC cleavage site. The portion of the substrate sequence that is depicted as a horizontal line is the region of homologous sequence of the holliday linker. Because the 5' end of the corresponding sequence in the substrate is provided with a 6-FAM fluorescent group, the enzyme digestion product of the sequence can be detected by a fluorescent glue scanner with corresponding wavelength after being separated by 15% TBE urea glue. Among them, ec ruvc is a recombinant ec ruvc protein sold by abcam corporation as a control. The leftmost lane of the gel is a single-stranded DNAmarker. The position marked by the arrow in the substrate is the cleavage site judged from the glue pattern, wherein the DrRuvC cleavage site is after TC, and the control EcRuvC cleavage site is after TT.

FIG. 6 is a screen for DrRuvC-favored Holiday linker junction region sequences. Four variable bases (N) ¹ N ² N ³ N ⁴ ) After cleavage of the enzyme with 10% TBE activated gel, the gel was stained with Stains-all dye (Bio-technology), and the results showed that DrRuvC preferentially dissociates the holliday linker containing 5'- (G/C) TC (G/C) -3' sequence at the junction.

FIG. 7 is a screening result of the optimal catalytic metal ions and concentrations in optimizing DrRuvC enzyme activity conditions. (A) DrRuvC at 10mM different metal ions (MgCl ₂ 、MnCl ₂ 、CaCl ₂ 、ZnSO ₄ 、NiCl ₂ 、CoCl ₂ And CuCl ₂ ) Comparison of catalytic efficiency under conditions.(B) DrRuvC is MgCl at various concentrations (0.31, 0.625, 1.25, 2.5, 5, 10, 20 mM) ₂ And MnCl ₂ Comparison of catalytic efficiency under conditions.

FIG. 8 is the result of screening for optimal pH in optimizing DrRuvC enzymatic activity conditions. Comparison of the catalytic efficiency of DrRuvC at different pH conditions (pH 5.6, 6.0, 6.4, 6.8, 7.2, 7.6, 8.0, 8.4, 8.8, 9.2).

FIG. 9 is a screening result of the optimal NaCl concentration in optimizing the DrRuvC enzyme activity condition. Comparison of catalytic efficiency of DrRuvC at different NaCl concentrations (50, 100, 150, 200, 250, 300, 350, 400, 450, 500 mM).

FIG. 10 is a screening result of the optimal reaction temperature in optimizing DrRuvC enzyme activity conditions. Comparison of the catalytic efficiency of DrRuvC at different temperatures (15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃,50 ℃, 55 ℃, 60 ℃).

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more apparent, the present invention will be further described in detail with reference to the drawings and examples of the specification. (the DNA sequences according to the examples were all synthesized by Shanghai Jieli, and the reagents and drugs according to the examples were purchased from Shanghai Biotechnology, if not explicitly stated

EXAMPLE 1 preparation of DrRuvC protein

1. Construction of DrRuvC prokaryotic expression vector:

the gene encoding DrRuvC was amplified directly from the genome of P.radiodurans using the primers DrRuvC-FN (5'-TACTTCCAAGGTCATATGAGGGTTCTGGGGATTGACCC-3', SEQ NO. 1) and DrRuvC-RB (5 'GAGCTCGAATTCGGATCTCGCGCCGCAGCGG-3', SEQ NO. 2), after confirming the correct size of the amplified fragment by agarose gel detection (FIG. 1), the PCR fragment was recovered using a gel recovery kit (Shanghai JieRuy), the fragment was ligated to a linearized pET28T expression vector which had been treated with NdeI and BamHI restriction enzymes and recovered, the ligation product was transformed into DH 5. Alpha. Clone competent, single colony sequencing was taken on LB plates containing Kana resistance (final concentration 50 mg/L), and after correct sequencing, a recombinant expression vector T28T-drruvC (FIG. 2) was proposed, which was able to express 6 DrvC tags at the N-terminal of the heterologous E.coli strain.

The linearized pET28T plasmid is a plasmid modified based on a commercialized pET28a plasmid, the main modification is that the sequence 'GGCCTGGTGCCGCGCGGCAGC' behind the His tag of the original pET28a plasmid is replaced by 'GAGAACCTGTACTTCCAAGGT', the new sequence just codes a TEV enzyme cutting site 'ENLYFQG', the modified expression plasmid can be fused and expressed at the N end of a target protein to form a section of short peptide 'MGSSHHHHHHSSENLYFQGH', the short peptide is favorable for carrying out affinity chromatography purification on the target protein by utilizing a nickel column at the later stage, the size of the short peptide generally does not influence the corresponding enzyme activity, in addition, the short peptide can be cut off by utilizing TEV enzyme when necessary, only two 'GH' amino acids are reserved at the N end of the target protein, and redundant amino acids irrelevant to the activity of the target protein are reduced to the greatest extent.

The nucleotide sequence of the gene encoding DrRuvC is specifically as follows:

atgagggttctggggattgaccccggtctggcgaacctgggcctgggactggtcgaaggggatgtccggcgggccaagcacctgtaccacgtctgcctgaccaccgaaagcgcgtggctgatgccccggcggctgcaatacctgcacgaggaactgacccggctgctcaccgagtaccggcccgacgcggtggcgatcgaggaccagattctgcgccgacaggctgacgtggcgttcaaagtggggcaggcgttcggggtggtgcagctcgcctgtgcgcaggccggggtgccgattcacgcctacggccccatgcaggtcaagaagtcgctggtgggcacgggccgcgccgacaaggagcaggtcatctacatggtcaaggcgagcctgggtattcgcgagctgttcaacaaccacgccgccgacgcgctggctctggcgctgacccacctcgcgcacgcgcccatgcaggagcgcagcgagcggctggcggcggcgggcagggctgcgcgcacaggagacgccccgctgcggcgctga, SEQ NO.3.

2. Heterologous expression of DrRuvC protein: the expression vector pET28T-drruvC was transformed into E.coli expression strain Rosetta (DE 3) and plated on LB plates containing Kana resistance (final concentration 50 mg/L) and chloramphenicol (final concentration 30 mg/L). Single colony is picked up and inoculated into 5ml of LB liquid medium, the culture is cultured overnight at 37 ℃, the culture is transferred into 1LLB liquid medium for expansion culture, when the OD of the bacterial liquid reaches 0.8, 0.8mM isopropyl-beta-D-thiogalactoside is added into the culture medium, meanwhile, the bacterial liquid is transferred to 18 ℃ for overnight culture (the target protein is expressed in a large amount at the step), then bacterial is collected, and the supernatant is discarded.

3. Purification of DrRuvC protein: the cells obtained in step 2 were suspended in a lysate (20 mM Tris [ pH8.0], 1M NaCl, 0.5mM Tris [ 2-carboxyyl ] phosphine [ TCEP ] and 5mM imidazole), lysed by a pressure breaker, and then centrifuged at 20000g at 4℃for 60 minutes to collect the supernatant. The supernatant was filtered with a 0.22 μm filter membrane, and then, a nickel column (nickel column A equilibrium, 6% nickel column B wash out the impurity protein, 100% nickel column B wash out), a desalting column (desalting A equilibrium and elution), a heparin sulfate column (heparin sulfate A equilibrium, 0-50% heparin sulfate B gradient elution) and a molecular sieve column (molecular sieve A equilibrium and elution) were sequentially used to obtain a very pure target protein. As described above, 1mg of DrRuvC protein with a purity of 99% or more can be finally obtained per liter of the bacterial culture. The purity of the final DrRuvC protein was determined by 15% SDS-PAGE (FIG. 3B). The purified DrRuvC molecular sieve (Superdex 75) pattern showed a peak position of 10ml, corresponding molecular weight of about 40-45kDa, and a molecular weight of about 20kDa for the DrRuvC monomer, demonstrating that DrRuvC exists in solution as homodimers (FIG. 3A). The buffer formulation used for the purification process was as follows:

(1) Nickel column a solution: 20mM Tris (pH 8.0), 1M NaCl, 0.5mM TCEP and 5mM imidazole; nickel column B solution: 20mM Tris (pH 8.0), 1M NaCl, 0.5mM TCEP and 500mM imidazole.

(2) Desalting solution A: 20mM Tris (pH 8.0), 100mM NaCl, 0.5mM TCEP.

(3) Heparin sulfate A solution: 20mM Tris (pH 8.0), 100mM NaCl, 0.5mM TCEP; heparin sulfate solution B: 20mM Tris (pH 8.0), 2M NaCl, 0.5mM TCEP.

(4) Molecular sieve A solution: 20mM Tris (pH 8.0), 100mM NaCl, 0.5mM TCEP.

The DrRuvC protein expressed in the engineering bacteria has the following amino acid sequence:

MGSSHHHHHHSSENLYFQGHMRVLGIDPGLANLGLGLVEGDVRRAKHLYHVCLTTESAWLMPRRLQYLHEELTRLLTEYRPDAVAIEDQILRRQADVAFKVGQAFGVVQLACAQAGVPIHAYGPMQVKKSLVGTGRADKEQVIYMVKASLGIRELFNNHAADALALALTHLAHAPMQERSERLAAAGRAARTGDAPLRR, SEQ NO.4.

Example 2 detection of DrRuvC Activity and screening of optimal substrates

1. The following sequence anneals were used to form an artificially synthesized holliday linker substrate containing a 66nt homologous sequence region for detection of DrRuvC activity (see fig. 4). Wherein FAM-J98-1, J98-2, J98-3 and J98-4 can anneal to form a holliday linker substrate HJ98a, and J98-1, FAM-J98-2, J98-3 and J98-4 can anneal to form a holliday linker substrate HJ98b, the portion of the sequence that is depicted as a horizontal line being a region of homologous sequence of the holliday linker.

The annealing conditions were: the sequences were mixed in equal proportions, heated to 95℃in 20mM Tris (pH 8.0), 50mM NaCl and 0.5mM TCEP buffer, and cooled slowly to room temperature over 2 hours.

The reaction process of the dissociation of the holliday linker is as follows: 10 μl of the reaction system contains 1 μM DrRuvC protein (or recombinant EcRuvC protein sold by abcam corporation as control), 500nM of holliday linker substrate HJ98a or HJ98b, 100mM NaCl, 10mM MgCl ₂ Or MnCl ₂ 20mM Tris (pH 8.0), 0.5mM TCEP, 5% glycerol. The reaction was allowed to stand at 37℃for 30 minutes, then was quenched by the addition of an equal volume of stop solution A (containing 20mM EDTA and 98% formamide), heated at 100℃for 20 minutes, and immediately transferred to ice quenching.

Because the 5' end of the corresponding sequence in the substrate is provided with a 6-FAM fluorescent group, the enzyme digestion product of the sequence can be separated by 15% TBE urea gum (200V constant pressure for 40 minutes) and detected by a fluorescent gum-sweeping instrument with corresponding wavelength. The leftmost lane of the gel is a single-stranded DNAmarker. In addition, ec ruvc is a recombinant ec ruvc protein sold by abcam corporation (cat No. P0a 814) as a control.

The final reaction results showed that DrRuvC was different from EcRuvC in the cleavage sites for HJ98a and HJ98b, with DrRuvC cleavage sites being mainly near sequences containing "TC" and EcRuvC cleavage sites being mainly near sequences containing "TT" (see fig. 4). It can be seen that DrRuvC is a novel holliday linker dissociating enzyme with a different activity than ectruvc.

2. Based on the results of step 1, a shorter holliday linker substrate HJ31 was designed to more precisely locate the cleavage site of DrRuvC. The holliday linker substrate HJ31 (see fig. 5) was formed by co-annealing FAM-J31-1, J31-2, J31-3 and J31-4 in the same proportions. The sequence involved is as follows:

The reaction process of the dissociation of the holliday linker is as follows: 10 μl of the reaction system contains 1 μM DrRuvC protein (or recombinant EcRuvC protein sold by abcam as control), 500nM of holliday linker substrate HJ31, 100mM NaCl, 10mM MnCl ₂ 20mM Tris (pH 8.0), 0.5mM TCEP, 5% glycerol. The reaction was allowed to stand at 37℃for 30 minutes, then was quenched by the addition of an equal volume of a stop solution A (containing 20mM EDTA and 98% formamide), heated at 100℃for 20 minutes, immediately transferred to ice for quenching, and then the product was isolated by means of 15% TBE urea gum (200V constant pressure for 50 minutes) and detected by means of a fluorescent gum-scanner of the corresponding wavelength. The leftmost lane of the gel is a single-stranded DNA marker. In addition, ec ruvc served as a control.

The final reaction results showed that DrRuvC was different from EcRuvC on the cleavage site of HJ31, drRuvC was mainly breaking the phosphodiester bond after cytosine in "TC", and EcRuvC was mainly breaking the phosphodiester bond after thymine in "TT" (see fig. 5). Further described is that DrRuvC is a novel holliday linker dissociating enzyme with a different activity than ectruvc.

3. Based on the results of steps 1 and 2, different bases were designed around "TC" to more precisely determine the DrRuvC-favored Holidar linker substrate sequence. In J24-N ¹ N ² N ³ N ⁴ -1、24-D ¹ D ² D ³ D ⁴ -2、24-N ¹ N ² N ³ N ⁴ -3 and 24-D ¹ D ² D ³ D ⁴ -4 (wherein N ¹ N ² N ³ N ⁴ And D ¹ D ² D ³ D ⁴ Is a variable base) and the like, can form a holliday linker substrate HJ24-N ¹ N ² N ³ N ⁴ (see FIG. 6). The sequence involved is as follows:

The reaction process of the dissociation of the holliday linker is as follows: 10. Mu.l of the reaction system contained 5. Mu.M of DrRuvC protein (or recombinant EcRuvC protein sold by abcam Co., ltd.) as a control, 2.5. Mu.M of the holliday linker substrate HJ24-N ¹ N ² N ³ N ⁴ 、100mM NaCl、10mM MnCl ₂ 20mM Tris (pH 8.0), 0.5mM TCEP, 5% glycerol. After reaction at 37℃for 30 minutes, the reaction was stopped by adding an equal volume of stop solution B (containing 20mM EDTA and 10mg/mL proteinase K), and after stopping the reaction at 37℃for 20 minutes, the product was isolated with 10% TBE-active gel (100V constant pressure for 50 minutes) and the gel was stained with Stains-all dye (Bio-technology) and photographed.

The final reaction results show that DrRuvC prefers to dissociate holliday linkers containing 5'- (G/C) TC (G/C) -3' sequences at the junction (see fig. 6).

Example 3: drRuvC enzyme activity condition optimization

1. Screening of DrRuvC optimum catalytic metal ions and concentration. The optimal catalytic metal ions and concentrations of DrRuvC were screened using the holliday linker HJ31 of example 2 as a substrate.

Wherein the screening process of the optimal metal ions for the enzyme activity reaction comprises the following steps: mu.l of the reaction system contained 1. Mu.M of DrRuvC protein, 500nM of the Holiday linker substrate HJ31, 100mM NaCl, 10mM metal ion (MgCl) ₂ 、MnCl ₂ 、CaCl ₂ 、ZnSO ₄ 、NiCl ₂ 、CoCl ₂ And CuCl ₂ ) 20mM Tris (pH 8.0), 0.5mM TCEP, 5% glycerol. The reaction was allowed to stand at 37℃for 30 minutes, then was quenched by the addition of an equal volume of a stop solution A (containing 20mM EDTA and 98% formamide), heated at 100℃for 20 minutes, immediately transferred to ice for quenching, and then the product was isolated by means of 15% TBE urea gum (200V constant pressure for 40 minutes) and detected by means of a fluorescent gum-scanner of the corresponding wavelength. The scan results were subjected to a banding analysis using ImageJ software (National Institutes of Health, USA) to determine the ratio of product and substrate bands after catalytic dissociation of each metal ion, respectively, and then a bar graph of dissociation efficiency of each metal ion under the reaction conditions was drawn using GraphPad Prism9 (San Diego, USA). Finally, mn ions and Mg ions are found to have better catalytic effect, and other metals have little activity (see FIG. 7A).

Wherein the metal ion concentration optimization process is as follows: 10 μl of the reaction system contains 1 μM DrRuvC protein, 500nM of the Holiday linker substrate HJ31, 100mM NaCl, different concentrations of MgCl ₂ Or MnCl ₂ (0.31, 0.625, 1.25, 2.5, 5, 10, 20 mM), 20mM Tris (pH 8.0), 0.5mM TCEP, 5% glycerol. The reaction was allowed to stand at 37℃for 30 minutes, then was quenched by the addition of an equal volume of a stop solution A (containing 20mM EDTA and 98% formamide), heated at 100℃for 20 minutes, immediately transferred to ice for quenching, and then the product was isolated by means of 15% TBE urea gum (200V constant pressure for 40 minutes) and detected by means of a fluorescent gum-scanner of the corresponding wavelength. The scan results were subjected to banding analysis using ImageJ software to determine the proportion of product and substrate bands for each lane, respectively, followed by plotting lines of dissociation efficiency at different metal ion concentrations using GraphPad Prism 9. It was finally found that DrRuvC has the strongest enzyme activity at Mn ion concentrations of 2.5-10mM and the strongest enzyme activity at Mg ion concentrations of 20mM. However, in general, the catalytic efficiency of Mn ions isStronger than Mg ion, is the best catalytic metal ion for DrRuvC (see fig. 7B).

2. Screening of optimal pH for DrRuvC reaction conditions. The process is as follows: mu.l of the reaction system contains 1. Mu.M of DrRuvC protein, 500nM of the Holiday linker substrate HJ31, 100mM NaCl, 10mM MnCl ₂ 50mM buffers of different pH (wherein buffers of pH 5.6, 6.0, 6.4, 6.8, 7.2 are prepared from Bis-Tris and hydrochloric acid, and buffers of pH7.6, 8.0, 8.4, 8.8, 9.2 are prepared from Tris and hydrochloric acid), 0.5mM TCEP, 5% glycerol. The reaction was allowed to stand at 37℃for 30 minutes, then was quenched by the addition of an equal volume of a stop solution A (containing 20mM EDTA and 98% formamide), heated at 100℃for 20 minutes, immediately transferred to ice for quenching, and then the product was isolated by means of 15% TBE urea gum (200V constant pressure for 40 minutes) and detected by means of a fluorescent gum-scanner of the corresponding wavelength. The scan results were subjected to banding analysis using ImageJ software to determine the proportion of product and substrate bands for each lane, respectively, followed by plotting lines of dissociation efficiency at different metal ion concentrations using GraphPadPrism 9. Finally, drRuvC was found to have a strong activity in the pH range of 7.6-8.4, with the strongest enzyme activity at pH8.0 (see FIG. 8).

3. Screening of optimal NaCl concentration for DrRuvC reaction conditions. The process is as follows: 10 μl of the reaction system contains 1 μM of DrRuvC protein, 500nM of the hollydi linker substrate HJ31, 10mM MnCl ₂ 20mM Tris (pH 8.0), naCl at different concentrations (final concentrations 50, 100, 150, 200, 250, 300, 350, 400, 450, 500mM respectively), 0.5mM TCEP, 5% glycerol. The reaction was allowed to stand at 37℃for 30 minutes, then was quenched by the addition of an equal volume of a stop solution A (containing 20mM EDTA and 98% formamide), heated at 100℃for 20 minutes, immediately transferred to ice for quenching, and then the product was isolated by means of 15% TBE urea gum (200V constant pressure for 40 minutes) and detected by means of a fluorescent gum-scanner of the corresponding wavelength. The scan results were subjected to banding analysis using ImageJ software to determine the proportion of product and substrate bands for each lane, respectively, followed by plotting lines of dissociation efficiency at different metal ion concentrations using GraphPad Prism 9. Finally, drRuvC was found to have the strongest activity at NaCl final concentrations ranging from 50 to 150mM (see FIG. 9).

4. Optimum DrRuvC reaction conditionsAnd (5) screening reaction temperature. 10 μl of the reaction system contains 1 μM of DrRuvC protein, 500nM of the hollydi linker substrate HJ31, 10mM MnCl ₂ 20mM Tris (pH 8.0), 100mM NaCl, 0.5mM TCEP, 5% glycerol. The reaction was stopped by allowing to stand at different temperatures (15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃,50 ℃, 55 ℃, 60 ℃) for 30 minutes, then adding an equal volume of stop solution A (containing 20mM EDTA and 98% formamide), heating at 100 ℃ for 20 minutes, immediately transferring to ice for quenching, then separating the product by using 15% TBE urea gum (200V constant pressure for 40 minutes), and detecting by using a fluorescent gum scanner of corresponding wavelength. The scan results were subjected to banding analysis using ImageJ software to determine the proportion of product and substrate bands for each lane, respectively, followed by plotting lines of dissociation efficiency at different metal ion concentrations using GraphPad Prism 9. Finally, drRuvC was found to be the most active at a reaction temperature in the range of 40-45℃ (see FIG. 10).

Sequence listing

<110> Hangzhou university of education

<120> a holliday connector dissociating enzyme DrRuvC, and encoding gene and application thereof

<160> 78

<170> SIPOSequenceListing 1.0

<210> 1

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

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tacttccaag gtcatatgag ggttctgggg attgaccc 38

<210> 2

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

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gagctcgaat tcggatcctc agcgccgcag cggg 34

<210> 3

<211> 540

<212> DNA

<213> Artificial sequence (Artificial Sequence)

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atgagggttc tggggattga ccccggtctg gcgaacctgg gcctgggact ggtcgaaggg 60

gatgtccggc gggccaagca cctgtaccac gtctgcctga ccaccgaaag cgcgtggctg 120

atgccccggc ggctgcaata cctgcacgag gaactgaccc ggctgctcac cgagtaccgg 180

cccgacgcgg tggcgatcga ggaccagatt ctgcgccgac aggctgacgt ggcgttcaaa 240

gtggggcagg cgttcggggt ggtgcagctc gcctgtgcgc aggccggggt gccgattcac 300

gcctacggcc ccatgcaggt caagaagtcg ctggtgggca cgggccgcgc cgacaaggag 360

caggtcatct acatggtcaa ggcgagcctg ggtattcgcg agctgttcaa caaccacgcc 420

gccgacgcgc tggctctggc gctgacccac ctcgcgcacg cgcccatgca ggagcgcagc 480

gagcggctgg cggcggcggg cagggctgcg cgcacaggag acgccccgct gcggcgctga 540

<210> 4

<211> 199

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Met Gly Ser Ser His His His His His His Ser Ser Glu Asn Leu Tyr

1 5 10 15

Phe Gln Gly His Met Arg Val Leu Gly Ile Asp Pro Gly Leu Ala Asn

20 25 30

Leu Gly Leu Gly Leu Val Glu Gly Asp Val Arg Arg Ala Lys His Leu

35 40 45

Tyr His Val Cys Leu Thr Thr Glu Ser Ala Trp Leu Met Pro Arg Arg

50 55 60

Leu Gln Tyr Leu His Glu Glu Leu Thr Arg Leu Leu Thr Glu Tyr Arg

65 70 75 80

Pro Asp Ala Val Ala Ile Glu Asp Gln Ile Leu Arg Arg Gln Ala Asp

85 90 95

Val Ala Phe Lys Val Gly Gln Ala Phe Gly Val Val Gln Leu Ala Cys

100 105 110

Ala Gln Ala Gly Val Pro Ile His Ala Tyr Gly Pro Met Gln Val Lys

115 120 125

Lys Ser Leu Val Gly Thr Gly Arg Ala Asp Lys Glu Gln Val Ile Tyr

130 135 140

Met Val Lys Ala Ser Leu Gly Ile Arg Glu Leu Phe Asn Asn His Ala

145 150 155 160

Ala Asp Ala Leu Ala Leu Ala Leu Thr His Leu Ala His Ala Pro Met

165 170 175

Gln Glu Arg Ser Glu Arg Leu Ala Ala Ala Gly Arg Ala Ala Arg Thr

180 185 190

Gly Asp Ala Pro Leu Arg Arg

195

<210> 5

<211> 98

<212> DNA

<213> FAM-J98-1

<400> 5

ttctaagacc ctgaaccact cgggaaataa caagatttca tctatgacca gtacgagctt 60

aggttgtcct ggcccgcgtg caaaggatga cagaagca 98

<210> 6

<211> 98

<212> DNA

<213> J98-1

<400> 6

ttctaagacc ctgaaccact cgggaaataa caagatttca tctatgacca gtacgagctt 60

aggttgtcct ggcccgcgtg caaaggatga cagaagca 98

<210> 7

<211> 98

<212> DNA

<213> FAM-J98-2

<400> 7

tgcttctgtc atcctttgca cgcgggccag gacaacctaa gctcgtactg gtcatagatg 60

aaatcttgtt atttcccgag tgtcaatcca tacttcgt 98

<210> 8

<211> 98

<212> DNA

<213> J98-2

<400> 8

tgcttctgtc atcctttgca cgcgggccag gacaacctaa gctcgtactg gtcatagatg 60

aaatcttgtt atttcccgag tgtcaatcca tacttcgt 98

<210> 9

<211> 98

<212> DNA

<213> J98-3

<400> 9

acgaagtatg gattgacact cgggaaataa caagatttca tctatgacca gtacgagctt 60

aggttgtcct ggcccgcgtg cagattctca agatgact 98

<210> 10

<211> 98

<212> DNA

<213> J98-4

<400> 10

agtcatcttg agaatctgca cgcgggccag gacaacctaa gctcgtactg gtcatagatg 60

aaatcttgtt atttcccgag tggttcaggg tcttagaa 98

<210> 11

<211> 31

<212> DNA

<213> FAM-J31-1

<400> 11

gctagccaca gccattcgga cgattgcggg a 31

<210> 12

<211> 31

<212> DNA

<213> J31-2

<400> 12

tcccgcaatc gtccgaaacc gagcacgatc t 31

<210> 13

<211> 31

<212> DNA

<213> J31-3

<400> 13

agatcgtgct cggtttccca ccagatgcca t 31

<210> 14

<211> 31

<212> DNA

<213> J31-4

<400> 14

atggcatctg gtccgaatgg ctgtggctag c 31

<210> 15

<211> 24

<212> DNA

<213> J24-TTCG-1

<400> 15

gccacagcca ttcgcccatt gcgt 24

<210> 16

<211> 24

<212> DNA

<213> J24-TTCG-2

<400> 16

acgcaatggg cgaaaccgag cacg 24

<210> 17

<211> 24

<212> DNA

<213> J24-TTCG-3

<400> 17

cgtgctcggt ttcgtgcaga tgcc 24

<210> 18

<211> 24

<212> DNA

<213> J24-TTCG-4

<400> 18

ggcatctgca cgaatggctg tggc 24

<210> 19

<211> 24

<212> DNA

<213> J24-ATCG-1

<400> 19

gccacagcca atcgcccatt gcgt 24

<210> 20

<211> 24

<212> DNA

<213> J24-ATCG-2

<400> 20

acgcaatggg cgataccgag cacg 24

<210> 21

<211> 24

<212> DNA

<213> J24-ATCG-3

<400> 21

cgtgctcggt atcgtgcaga tgcc 24

<210> 22

<211> 24

<212> DNA

<213> J24-ATCG-4

<400> 22

ggcatctgca cgattggctg tggc 24

<210> 23

<211> 24

<212> DNA

<213> J24-CTCG-1

<400> 23

gccacagcca ctcgcccatt gcgt 24

<210> 24

<211> 24

<212> DNA

<213> J24-CTCG-2

<400> 24

acgcaatggg cgagaccgag cacg 24

<210> 25

<211> 24

<212> DNA

<213> J24-CTCG-3

<400> 25

cgtgctcggt ctcgtgcaga tgcc 24

<210> 26

<211> 24

<212> DNA

<213> J24-CTCG-4

<400> 26

ggcatctgca cgagtggctg tggc 24

<210> 27

<211> 24

<212> DNA

<213> J24-GTCG-1

<400> 27

gccacagcca gtcgcccatt gcgt 24

<210> 28

<211> 24

<212> DNA

<213> J24-GTCG-2

<400> 28

acgcaatggg cgacaccgag cacg 24

<210> 29

<211> 24

<212> DNA

<213> J24-GTCG-3

<400> 29

cgtgctcggt gtcgtgcaga tgcc 24

<210> 30

<211> 24

<212> DNA

<213> J24-GTCG-4

<400> 30

ggcatctgca cgactggctg tggc 24

<210> 31

<211> 24

<212> DNA

<213> J24-TTCC-1

<400> 31

gccacagcca ttcccccatt gcgt 24

<210> 32

<211> 24

<212> DNA

<213> J24-TTCC-2

<400> 32

acgcaatggg ggaaaccgag cacg 24

<210> 33

<211> 24

<212> DNA

<213> J24-TTCC-3

<400> 33

cgtgctcggt ttcctgcaga tgcc 24

<210> 34

<211> 24

<212> DNA

<213> J24-TTCC-4

<400> 34

ggcatctgca ggaatggctg tggc 24

<210> 35

<211> 24

<212> DNA

<213> J24-ATCC-1

<400> 35

gccacagcca atcccccatt gcgt 24

<210> 36

<211> 24

<212> DNA

<213> J24-ATCC-2

<400> 36

acgcaatggg ggataccgag cacg 24

<210> 37

<211> 24

<212> DNA

<213> J24-ATCC-3

<400> 37

cgtgctcggt atcctgcaga tgcc 24

<210> 38

<211> 24

<212> DNA

<213> J24-ATCC-4

<400> 38

ggcatctgca ggattggctg tggc 24

<210> 39

<211> 24

<212> DNA

<213> J24-CTCC-1

<400> 39

gccacagcca ctcccccatt gcgt 24

<210> 40

<211> 24

<212> DNA

<213> J24-CTCC-2

<400> 40

acgcaatggg ggagaccgag cacg 24

<210> 41

<211> 24

<212> DNA

<213> J24-CTCC-3

<400> 41

cgtgctcggt ctcctgcaga tgcc 24

<210> 42

<211> 24

<212> DNA

<213> J24-CTCC-4

<400> 42

ggcatctgca ggagtggctg tggc 24

<210> 43

<211> 24

<212> DNA

<213> J24-GTCC-1

<400> 43

gccacagcca gtcccccatt gcgt 24

<210> 44

<211> 24

<212> DNA

<213> J24-GTCC-2

<400> 44

acgcaatggg ggacaccgag cacg 24

<210> 45

<211> 24

<212> DNA

<213> J24-GTCC-3

<400> 45

cgtgctcggt gtcctgcaga tgcc 24

<210> 46

<211> 24

<212> DNA

<213> J24-GTCC-4

<400> 46

ggcatctgca ggactggctg tggc 24

<210> 47

<211> 24

<212> DNA

<213> J24-TTCA-1

<400> 47

gccacagcca ttcacccatt gcgt 24

<210> 48

<211> 24

<212> DNA

<213> J24-TTCA-2

<400> 48

acgcaatggg tgaaaccgag cacg 24

<210> 49

<211> 24

<212> DNA

<213> J24-TTCA-3

<400> 49

cgtgctcggt ttcatgcaga tgcc 24

<210> 50

<211> 24

<212> DNA

<213> J24-TTCA-4

<400> 50

ggcatctgca tgaatggctg tggc 24

<210> 51

<211> 24

<212> DNA

<213> J24-ATCA-1

<400> 51

gccacagcca atcacccatt gcgt 24

<210> 52

<211> 24

<212> DNA

<213> J24-ATCA-2

<400> 52

acgcaatggg tgataccgag cacg 24

<210> 53

<211> 24

<212> DNA

<213> J24-ATCA-3

<400> 53

cgtgctcggt atcatgcaga tgcc 24

<210> 54

<211> 24

<212> DNA

<213> J24-ATCA-4

<400> 54

ggcatctgca tgattggctg tggc 24

<210> 55

<211> 24

<212> DNA

<213> J24-CTCA-1

<400> 55

gccacagcca ctcacccatt gcgt 24

<210> 56

<211> 24

<212> DNA

<213> J24-CTCA-2

<400> 56

acgcaatggg tgagaccgag cacg 24

<210> 57

<211> 24

<212> DNA

<213> J24-CTCA-3

<400> 57

cgtgctcggt ctcatgcaga tgcc 24

<210> 58

<211> 24

<212> DNA

<213> J24-CTCA-4

<400> 58

ggcatctgca tgagtggctg tggc 24

<210> 59

<211> 24

<212> DNA

<213> J24-GTCA-1

<400> 59

gccacagcca gtcacccatt gcgt 24

<210> 60

<211> 24

<212> DNA

<213> J24-GTCA-2

<400> 60

acgcaatggg tgacaccgag cacg 24

<210> 61

<211> 24

<212> DNA

<213> J24-GTCA-3

<400> 61

cgtgctcggt gtcatgcaga tgcc 24

<210> 62

<211> 24

<212> DNA

<213> J24-GTCA-4

<400> 62

ggcatctgca tgactggctg tggc 24

<210> 63

<211> 24

<212> DNA

<213> J24-TTCT-1

<400> 63

gccacagcca ttctcccatt gcgt 24

<210> 64

<211> 24

<212> DNA

<213> J24-TTCT-2

<400> 64

acgcaatggg agaaaccgag cacg 24

<210> 65

<211> 24

<212> DNA

<213> J24-TTCT-3

<400> 65

cgtgctcggt ttcttgcaga tgcc 24

<210> 66

<211> 24

<212> DNA

<213> J24-TTCT-4

<400> 66

ggcatctgca agaatggctg tggc 24

<210> 67

<211> 24

<212> DNA

<213> J24-ATCT-1

<400> 67

gccacagcca atctcccatt gcgt 24

<210> 68

<211> 24

<212> DNA

<213> J24-ATCT-2

<400> 68

acgcaatggg agataccgag cacg 24

<210> 69

<211> 24

<212> DNA

<213> J24-ATCT-3

<400> 69

cgtgctcggt atcttgcaga tgcc 24

<210> 70

<211> 24

<212> DNA

<213> J24-ATCT-4

<400> 70

ggcatctgca agattggctg tggc 24

<210> 71

<211> 24

<212> DNA

<213> J24-CTCT-1

<400> 71

gccacagcca ctctcccatt gcgt 24

<210> 72

<211> 24

<212> DNA

<213> J24-CTCT-2

<400> 72

acgcaatggg agagaccgag cacg 24

<210> 73

<211> 24

<212> DNA

<213> J24-CTCT-3

<400> 73

cgtgctcggt ctcttgcaga tgcc 24

<210> 74

<211> 24

<212> DNA

<213> J24-CTCT-4

<400> 74

ggcatctgca agagtggctg tggc 24

<210> 75

<211> 24

<212> DNA

<213> J24-GTCT-1

<400> 75

gccacagcca gtctcccatt gcgt 24

<210> 76

<211> 24

<212> DNA

<213> J24-GTCT-2

<400> 76

acgcaatggg agacaccgag cacg 24

<210> 77

<211> 24

<212> DNA

<213> J24-GTCT-3

<400> 77

cgtgctcggt gtcttgcaga tgcc 24

<210> 78

<211> 24

<212> DNA

<213> J24-GTCT-4

<400> 78

ggcatctgca agactggctg tggc 24

Claims

1. A method for dissociating a Holiday connector is characterized in that 10 mul of a reaction system is placed at 40-45 ℃ for 30 minutes, then an equal volume of stop solution A is added for stopping the reaction, heating is carried out at 100 ℃ for 20 minutes, the mixture is transferred to ice for quenching, and then TBE urea gum is utilized for separating products; the stop solution A is 20mM EDTA and 98% formamide;

the 10 mu l reaction system contains 1 mu M of enzyme DrRuvC, holliday connector substrate containing 5'- (G/C) TC (G/C) -3' sequence at 500nM junction, 50-150mM NaCl, a certain concentration of metal ions, 20mM Tris with pH of 7.6-8.4, 0.5mM TCEP and 5% glycerol; the amino acid sequence of the enzyme DrRuvC is shown as SEQ NO.4;

the metal ion is Mn or Mg; when the metal ion is Mn, the concentration of Mn ions in the reaction system is 2.5-10 mM; when the metal ion is Mg, the concentration of the Mg ion in the reaction system is 20mM.

2. A method for dissociating a Holiday connector is characterized in that 10 mul of a reaction system is placed at 40-45 ℃ to react for 30 minutes, then an equal volume of stop solution B is added to stop the reaction, and after the reaction is stopped at 37 ℃ for 20 minutes, TBE active glue is utilized to separate products; the stop solution B is 20mM EDTA and 10mg/mL proteinase K;