DNA polymerase for improving synthesis efficiency of catalytic DNA

A technology of polymerase and ability, applied in the field of enzyme engineering, can solve the problem of short-term binding of Dbh to DNA

Active Publication Date: 2015-04-29
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But in order to prevent more mutations, Dbh will be excised immediately after spanning the lesion, and the normal replicative polymerase will resume the control of DNA synthesis, which shows that the binding of Dbh to DNA is short-lived

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  • DNA polymerase for improving synthesis efficiency of catalytic DNA
  • DNA polymerase for improving synthesis efficiency of catalytic DNA
  • DNA polymerase for improving synthesis efficiency of catalytic DNA

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Experimental program
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Effect test

Embodiment 1

[0018] Example 1 Determination of Mutation Sites

[0019] By comparing homologous sequences, the non-conserved sites in the Dbh sequence and the mutation direction and frequency of these sites were determined. The results are shown in Table 1. Eight mutation directions of T37F, I62V, M76I, A221S, Y249I, L250V, K337R and KSKIP (241-245) RVRKS were determined, and the binding and binding of these eight mutants Dbh and DNA were simulated by computer. Combined free energy calculations. The results are shown in Table 2. In theory, the reduction of binding free energy means that the binding of Dbh to the substrate is more stable, which means that the affinity between the enzyme and the substrate is greater, so that the continuous synthesis ability of the enzyme is improved. From the analysis in Table 2, it can be seen that , all mutations except K337R and Y249I can enhance the processivity.

[0020] Table 1. Mutation residue types and frequencies of non-conservative amino acids

...

Embodiment 2

[0026] Embodiment 2 Sdbh mutant enzyme construction and processivity comparison

[0027] Eight mutants Dbh T37F, Dbh I62V, Dbh M76I, DbhA221S, DbhKSKIP(241-245)RVRKS, Dbh Y249I, Dbh L250V, Dbh K337R were constructed by site-directed mutagenesis, and the N-terminal of each mutant was connected to the protein by a flexible linker Sso7d, 8 corresponding Sdbh mutants were obtained: Sdbh T37F, Sdbh I62V, Sdbh M76I, Sdbh A221S, Sdbh KSKIP(241-245)RVRKS, Sdbh Y249I, Sdbh L250V, Sdbh K337R. The nucleotide sequence encoding Dbh is shown in SEQ ID NO.1, and the starting amino acid sequence of Dbh is shown in SEQ ID NO.2, which encodes Dbh after the N-terminus is connected to the protein Sso7d through a flexible linker, that is, the nucleotide of Sdbh The sequence is shown in SEQ ID NO.3, the nucleotide sequence encoding the flexible linker is shown in SEQ ID NO.4, and the nucleotide sequence encoding Sso7d is shown in SEQ ID NO.5.

[0028] The processivity of Sdbh and its eight mutants...

Embodiment 3

[0031] The catalytic activity comparison of embodiment 3Sdbh and Sdbh mutant

[0032] 12.5nM of the annealed primer / template was added to the reaction buffer (10mM HEPES NaOH (pH7.4), 50mMNaCl, 10mM MgCl 2, 200 mM dNTPs, 1 mM DTT, 100 μg / ml BSA and 0.1% Triton X-100). 12.5nM DNA polymerase was added to initiate DNA synthesis at 37°C. At different time points, 1 μL samples were added to 99 μL PicoGreen (Molecular Probes) diluted 1:200, and reacted in TE buffer (10 mM Tris-Hcl pH 8.0 and 1 mM EDTA). The amount of synthesized DNA was quantified using an H1 hybrid multi-function microplate reader (Burten Instruments, USA). The unit activity of DNA polymerases was determined by comparing their initial rates with Sdbh.

[0033] The result is as figure 2 As shown, the Sdbh mutant exhibited an increase in polymerase activity compared to Sdbh, suggesting that mutations of non-conserved residues do not reduce polymerase activity but significantly increase the initial rate of DNA sy...

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Abstract

The invention discloses DNA polymerase for improving the synthesis efficiency of catalytic DNA and belongs to the technical field of biological engineering. The DNA polymerase is prepared by the following steps of: firstly mutating the 241-245th amino acids KSKIP of Dhb into RVRKS, or mutating the 250th-site L into V, or mutating the 221th-site A into S, or mutating the 76-site M into I, then fusing Sso7d by flexible linker at the N end of a mutant and constructing out Dbh with improvement of the nucleotide doping efficiency, i.e., four positive mutants such as SdhbM76I, Sdbh A221S, Sdbh KSKIP(241-245)RVRKS and Sdbh L250V. Simultaneously, the invention also provides a method for enhancing the continuous synthesis capability by non-conservative sites of a mutant enzyme.

Description

technical field [0001] The invention relates to a DNA polymerase with improved catalytic DNA synthesis efficiency, more specifically relates to a modified DNA polymerase with improved DNA affinity, belonging to the field of enzyme engineering. Background technique [0002] Y-family DNA polymerase Dbh is a kind of translesion synthetic polymerase (TLS), which can replace replicative DNA polymerase across template damage to continue DNA synthesis, thereby helping cells resist DNA damage. But in order to prevent more mutations, Dbh will be excised immediately after spanning the lesion, and the normal replicative polymerase will resume the control of DNA synthesis, which shows that the combination of Dbh and DNA is short-lived. The structure of Dbh is a typical right-handed structure, which is divided into four domains: thumb, palm, finger, and little-finger. Compared with other DNA polymerases, the finger domain of Dbh is very small , resulting in almost no contact with the ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54
CPCC12N9/1252C12Y207/07007
Inventor 吴静
Owner JIANGNAN UNIV
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