WO2018076980A1 - 一种催化dna合成延伸能力提高的dna聚合酶 - Google Patents

一种催化dna合成延伸能力提高的dna聚合酶 Download PDF

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WO2018076980A1
WO2018076980A1 PCT/CN2017/103118 CN2017103118W WO2018076980A1 WO 2018076980 A1 WO2018076980 A1 WO 2018076980A1 CN 2017103118 W CN2017103118 W CN 2017103118W WO 2018076980 A1 WO2018076980 A1 WO 2018076980A1
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dpo4
dna
dna polymerase
synthesis
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吴静
王立
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江南大学
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  • the invention relates to a DNA polymerase which catalyzes the improvement of DNA synthesis and extension ability, and belongs to the field of enzyme engineering.
  • the Y-family DNA polymerase Dpo4 is a type of trans-damage synthetic polymerase (TLS) that replaces replicative DNA polymerases across the template lesions to allow DNA synthesis to continue, thereby helping cells resist DNA damage.
  • TLS trans-damage synthetic polymerase
  • Dpo4 will be removed immediately after injury, and normal replicative polymerase will restore control of DNA synthesis, which requires the binding of Dpo4 to DNA to be transient.
  • Dpo4 is a typical Y-family DNA polymerase with a typical right-handed structure. It is divided into four domains: thumb, palm, finger, and little-finger. The enzyme, Dpo4 has a small finger field, resulting in almost no contact with the major groove of the nascent base pair.
  • Dpo4 In addition, its thumb domain is short and thick, making Dpo4 less active with DNA and incorporation of nucleosides. Dpo4 imposes very few constraints on its DNA substrate. Its structural characteristics and functional requirements determine Dpo4's low continuous synthesis capability.
  • the essence of sustained synthesis is to retain the affinity of the enzyme for the polymeric substrate in multiple rounds of catalysis. Therefore, increasing the affinity of the polymerase for the substrate is the essential way to improve the ability to continue synthesis.
  • the ability to increase polymerase extension in existing studies is primarily through the attachment of the corresponding binding proteins on the polymerase, such as the beta-sliding clip, thioredoxin, PCNA, and the Sso7d protein from Sulfolobus solfataricus.
  • Studies on enhancing the affinity of polymerases to DNA by mutating amino acids have been mainly demonstrated in the study of reverse transcriptases of some HIV viruses, and little research has been done on Y-family DNA polymerases.
  • the problem to be solved by the present invention is to provide a DNA polymerase Dpo4 with enhanced synthetic ability, which is mainly obtained by mutating the amino acid A of position 181 of Dpo4 to D by site-directed mutagenesis or by mutating the E at position 63 to K.
  • the sustained synthesis ability refers to the average length at which DNA polymerase binds once to extend DNA.
  • amino acid A at position 181 and the amino acid E at position 63 are all non-conserved sites.
  • the nucleotide sequence encoding wild type Dpo4 is shown in SEQ ID NO. 1, and the amino acid sequence of wild type Dpo4 is shown in SEQ ID NO.
  • a second technical problem to be solved by the present invention is to provide a method for obtaining the DNA polymerase Dpo4 with enhanced sustained synthesis ability, which is an experimental method for simulating the binding energy of Dpo4 and DNA by molecular dynamics means, and then site-directed mutagenesis. Mutants were obtained.
  • the present invention is based on wild type Dpo4 derived from Sulfolobus solfataricus, and the mutant is constructed to obtain DNA.
  • Dpo4 DNA polymerase
  • the elongation of Dpo4 A181D was increased by 25% compared with wild-type Dpo4
  • Dpo4 E63K was increased by 18.75% compared with Dpo4.
  • the fidelity of Dpo4 A181D and Dpo4 is close, and the fidelity of Dpo4 E63K is improved compared to Dpo4 and Dpo4 A181D.
  • the sustained synthesis ability of the mutants A181D and E63K obtained by the screening of the present invention is improved, and these modified Dpo4 and the kit carrying the enzyme will have an important positive effect on the operation of genetic engineering.
  • the recombinant plasmid pET28a-dpo4 was constructed by restriction enzyme ligation of the Dpo4 gene represented by SEQ ID NO. 1 by the biological company (Suzhou Xunxun), and transformed into E. coli BL21 strain, and pET28a contains histidine. After labeling, the expressed protein can be purified by Ni-NTA gravity column, then purified by cation exchange column Mono-S to homogeneity of the band, and SDS-PAGE is used to detect the purity of the target protein.
  • Non-conserved sites in the Dpo4 sequence and the direction and frequency of mutation of these sites were determined by homologous sequence alignment.
  • the results are shown in Table 1.
  • Ten mutation directions of F33Y, F37T, I59M, E63K, M76I, A181D, N188S, A220S, I248Y and V289I were determined, and the binding and binding freedom of the ten mutants Dpo4 and DNA were simulated by computer. Can calculate.
  • the results are shown in Table 2.
  • the combination of free energy reduction means that the binding of Dpo4 to the substrate is more stable, which means that the affinity of the enzyme to the substrate is greater, and the continuous synthesis ability of the enzyme is improved.
  • the other mutations can enhance the ability of continuous synthesis.
  • Dpo4 and its ten mutants were evaluated by primer extension experiments.
  • a constant concentration (10 nM) of annealed fluorescently labeled primers/templates was added to the reaction buffer (10 mM HEPES NaOH (pH 7.4), 50 mM NaCl, 10 mM MgCl 2 , 200 mM dNTPs, 1 mM DTT, 100 ⁇ g/ml BSA and 0.1% Triton X-100).
  • 100 nM of Dpo4 and its mutant enzyme initiate DNA synthesis at 37 ° C and use a 50-mer single-stranded sequence of 5000-fold excess of substrate as a Trap, controlling the enzyme to bind to DNA only once.
  • the continuous synthesis ability can be defined as the average extended strip length.
  • the fluorescence value according to the extended strip is the total extension.
  • Proportion, weighted average calculation of the average length of DNA polymerase binding to DNA extension, and as a measure of the ability to continue to synthesize, the wild-type Dpo4 has a sustained synthesis capacity of 16 nt, which is close to the existing literature data.
  • the enzyme is 20 times more than the DNA, it can extend a fragment of about 50-100, but it can be considered that the polymerase recombines due to the absence of Trap.
  • due to the presence of Trap and Dpo4 was only 10 times more than DNA, it was considered that the Dp4 sustained synthesis ability was 16 nt when the enzyme was 10-fold excess in DNA at 37 °C.
  • the average extended band length of all mutants was calculated according to the average length calculation method of wild-type Dpo4 (Fig. 2B), in which Dpo4A181D had the longest extension length, and the extension length was increased by 25% compared with wild-type Dpo4, and Dpo4E63K was compared with Dpo4.
  • the increase of 18.75% the extension length of Dpo4V289I, Dpo4A220S, Dpo4M76I, Dpo4F37T decreased compared with Dpo4, and the decrease of Dpo4V289I was 18.75%.
  • the remaining synthetic ability of the remaining mutants was close to that of wild-type Dpo4.
  • the results of the sustained synthesis ability of the mutant showed that some of the mutants with increased binding energy enhanced the sustained synthesis ability of Dpo4, and the mutants with weakened binding showed a lower sustained synthesis ability than wild-type Dpo4.
  • the Dpu4A181D, Dpo4E63K, and Dpo4V63I with the most sustained decrease in sustained ability were selected to compare the fidelity of Dpo4V289I with wild-type Dpo4 under Mn 2+ conditions.
  • Four templates (Table 3) were observed in single bases (dATP, dCTP, Primer extension under dGTP, dTTP) and mixed base dNTP conditions.
  • reaction buffer (10 mM HEPES NaOH (pH 7.4), 50 mM NaCl, 10 mM MnCl 2 , 200 mM dNTPs, 1 mM DTT, 100 ⁇ g/ml BSA and 0.1% Triton X-100).
  • DNA synthesis was initiated at 37 ° C by the addition of 10 nM DNA polymerase. After incubation for 2 h, the reaction was quenched with 10 ⁇ L of stop solution, and the reaction product was separated by 20% Urea-PAGE, and the gel was scanned by Typhoon Trio.
  • the incorporation rate of Dpo4 to dTTP was 88.48%, while that of Dpo4A181D was only 67.47. %, but the Dpo4A181D mismatch under the T G template is more serious than Dpo4.
  • the incorporation rate of Dpo4A181D into dATP, dTTP and dGTP is 73.27%, 60.95% and 63.07%, respectively, while the incorporation rate of Dpo4 is only 64.94. %, 59.52%, 46.67%.
  • Dpo4A181D is more serious than that of Dpo4, and the fidelity is lower than that of Dpo4.
  • Dpo4E63K only achieves 80% incorporation of dTTP under template T T , and the mismatching rate of other mismatches is lower than that. 60%.
  • the mismatch rate of Dpo4V289I was 67.58% in the case of incorporation of dGTP under the T A template, which was the maximum mismatch rate under the action of Dpo4V289I.
  • the fidelity of Dpo4A181D and Dpo4 is close, the fidelity of Dpo4E63K is improved compared with Dpo4 and Dpo4A181D, and the mismatch of Dpo4V289I is the best, the fidelity is the best, and the fidelity is high to low.
  • the order is: Dpo4V289I, Dpo4E63K, Dpo4, Dpo4A181D, indicating that the improvement of continuous synthesis ability is not necessarily related to the change of fidelity.
  • K obs K p [dNTP]/ ⁇ [dNTP]+K d,dNTP ⁇ calculates the maximum nucleotide incorporation rate K p and the dissociation constant K d, dNTP , to calculate the corresponding nucleotide incorporation efficiency (K p /K d, dNTP ) .
  • the nucleotide incorporation rate (K p ) of Dpo4A181D and Dpo4E63K was 1.8 times and 2.6 times that of Dpo4, respectively, but the affinity of Dpo4A181D and Dpo4E63K with nucleotides (K d ) was reduced by 1.7 compared with Dpo4 . 3 times, the nucleotide incorporation efficiency of the three is similar, and the nucleotide incorporation efficiency of Dpo4V289I is 45.9% lower than that of wild-type Dpo4. This lower nucleotide incorporation efficiency may determine Dpo4V289I has better fidelity than Dpo4 and other mutants.
  • Kd , dNTP reflects the affinity of the ground state binding for correct nucleotide incorporation, indicating the interaction of Dpo4 with adjacent base pairs.
  • K Dpo4A181D and Dpo4E63K both increased processivity mutant compared with the wild type Dpo4 d, weaker binding affinity, likely mutant increased processivity require "enzyme -DNA-dNTP" Faster From “open state” to "closed state", this process requires a conformational change between the finger and LF regions of Dpo4, which can weaken the interaction between dNTP and Dpo4, making the interaction between Dpo4 and the replicate base pair looser.
  • the K d value increases, and the low sustained synthesis ability of Dpo4V289I corresponds to a stronger ground state affinity.
  • nucleotide incorporation efficiency of Dpo4V289I was similar to that of Dpo4, and Dpo4A181D and Dpo4E63K had no effect on improving the continuous synthesis ability. Nucleotide incorporation efficiency.

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Abstract

一种催化DNA合成延伸能力提高的DNA聚合酶，是以来源于Sulfolobus solfataricus的野生型Dpo4为基础，构建突变体获得的DNA持续合成能力增强的DNA聚合酶Dpo4。Dpo4 A181D的延伸长度相比野生型Dpo4，增加了25％，Dpo4 E63K较Dpo4增加了18.75％。Dpo4 A181D和Dpo4的保真性接近，Dpo4 E63K的保真性相比Dpo4和Dpo4 A181D有所提高。所述突变体A181D、E63K的持续合成能力提高，这些改造后的Dpo4以及携带该酶的试剂盒将对基因工程的操作产生重要积极作用。

Description

一种催化DNA合成延伸能力提高的DNA聚合酶
A DNA Polymerase with Increased Ability to Extend DNA
技术领域
本发明涉及一种催化DNA合成延伸能力提高的DNA聚合酶，属于酶工程领域。
背景技术
Y-家族DNA聚合酶Dpo4是一类跨损伤合成聚合酶(TLS)，它能够代替复制性DNA聚合酶跨越模板损伤处使得DNA合成继续，从而帮助细胞抵抗DNA损害。但为了防止出现更多突变，跨越损伤后Dpo4会被立即切除，正常的复制性聚合酶会恢复对DNA合成的控制，这就要求Dpo4与DNA的结合是短暂的。Dpo4是典型的Y-家族DNA聚合酶，具有典型的右手结构，分为拇指(thumb)、手掌(palm)、手指(finger)、小手指(little-finger)四个结构域，相比其它DNA聚合酶，Dpo4的手指域很小，导致与新生碱基对的大沟几乎没有接触，另外它的拇指域短而粗，使得Dpo4与DNA及掺入核苷具有较少的作用，总的来说，Dpo4对其DNA底物施加的约束很少。它的结构特点和功能要求决定了Dpo4有着较低的持续合成能力。
持续性合成能力的本质是在多轮催化中保留酶对聚合底物的亲和性，因此，提高聚合酶对底物的亲和力才是提高持续合成能力的本质途径。在已有的研究中提高聚合酶延伸能力主要是通过在聚合酶上连接相应的结合蛋白，如β-滑动夹子，硫氧还蛋白，PCNA以及来自于Sulfolobus solfataricus的Sso7d蛋白。通过突变氨基酸来增强聚合酶与DNA的亲和力的研究主要在一些HIV病毒的逆转录酶的研究中有所体现，而关于Y-家族DNA聚合酶的研究很少。
发明内容
本发明要解决的问题是提供一种持续合成能力增强的DNA聚合酶Dpo4，主要是通过定点突变将Dpo4第181位的氨基酸A突变为D，或将第63位的E突变为K得到。
所述持续合成能力是指DNA聚合酶一次结合使DNA延伸的平均长度。
所述第181位氨基酸A、第63位的氨基酸E均为非保守位点。
编码野生型Dpo4的核苷酸序列如SEQ ID NO.1所示，野生型Dpo4的氨基酸序列如SEQ ID NO.2所示。
本发明要解决的第二个技术问题是提供一种获得所述持续合成能力增强的DNA聚合酶Dpo4的方法，是通过分子动力学手段理论模拟Dpo4与DNA的结合能，然后定点突变的实验方法获得突变体。
本发明以来源于Sulfolobus solfataricus的野生型Dpo4为基础，构建突变体获得DNA持 续合成能力增强的DNA聚合酶Dpo4。突变体的持续合成能力方面，Dpo4 A181D的延伸长度相比野生型Dpo4，增加了25％，Dpo4 E63K较Dpo4增加了18.75％。Dpo4 A181D和Dpo4的保真性接近，Dpo4 E63K的保真性相比Dpo4和Dpo4 A181D有所提高。总的来说，本发明筛选获得的突变体A181D、E63K的持续合成能力提高，这些改造后的Dpo4以及携带该酶的试剂盒将对基因工程的操作产生重要积极作用。
附图说明
图1Dpo4蛋白的表达及纯化，“1”代表蛋白Marker，“2”代表未经诱导的粗酶液，“3”代表经过IPTG诱导的粗酶液，“4”、“5”分别表示经过镍柱纯化和阳离子交换柱进一步纯化得到的蛋白。
图2Dpo4野生型酶与突变体酶的持续合成能力
图3Dpo4野生型酶及突变体的保真性比较
图4Dpo4野生型酶及突变体的核苷酸掺入效率比较
具体实施方式
实施例1Dpo4蛋白的表达和纯化
通过生物公司(苏州泓迅)合成核苷酸序列如SEQ ID NO.1所示的Dpo4目的基因，酶切连接构建重组质粒pET28a-dpo4，转化至E.coli BL21菌株表达，pET28a含有组氨酸标签，表达后蛋白可以通过Ni-NTA重力柱进行初步纯化，后利用阳离子交换柱Mono-S纯化至条带均一，并进行SDS-PAGE检测目的蛋白纯度。纯化后的Dpo4小样品-80℃保存在50mM Tris-HCl缓冲液中(pH 7.7 at 22℃)，该缓冲液含有50mM NaCl、1mM二硫苏糖醇和50％甘油(v/v)。蛋白表达及纯化的结果如图1所示，由条带2、3可以看出E.coli BL21在经过诱导后才会表达出Dpo4，不存在本底表达，由“5”可知，经过两步纯化后，目的蛋白以单一条带存在。
实施例2突变位点的确定
通过同源序列比对，确定了Dpo4序列中的非保守位点以及这些位点的突变方向及频率。结果如表1所示，确定了F33Y、F37T、I59M、E63K、M76I、A181D、N188S、A220S、I248Y、V289I共十个突变方向，利用计算机模拟这十个突变体Dpo4与DNA的结合及结合自由能计算。结果如表2所示，理论上结合自由能降低意味着Dpo4与底物的结合更为稳定，也就说明酶与底物的亲和力更大，从而酶的持续合成能力提高，从表2分析可知，除了F37T和A220S外其余突变都能增强持续合成能力。
表1.非保守氨基酸的突变残基种类及频率
Figure PCTCN2017103118-appb-000001
表2.Dpo4及其突变体与DNA复合物的结合自由能
Figure PCTCN2017103118-appb-000002
实施例3Dpo4突变酶构建及持续合成能力比较
通过定点突变分别构建10个突变体Dpo4F33Y、Dpo4F37T、Dpo4I59M、Dpo4E63K、Dpo4M76I、Dpo4A181D、Dpo4N188S、Dpo4A220S、Dpo4I248Y、Dpo4V289I，编码野生型Dpo4的核苷酸序列如SEQ ID NO.1所示，野生型Dpo4的氨基酸序列如SEQ ID NO.2所示。
通过引物延伸实验来评价Dpo4及其十个突变体的持续合成能力。恒定浓度(10nM)的退火好的荧光标记引物/模板添加到反应缓冲液(10mM HEPES NaOH(pH7.4)，50mM NaCl，10mM MgCl2，200mM dNTPs，1mM DTT，100μg/ml BSA和0.1％的Triton X-100)。100nM 的Dpo4及其突变体酶在37℃启动DNA的合成，并以5000倍过量于底物的50-mer单链序列作为Trap，控制酶只一次结合于DNA。孵育5min后，加入10μL终止液淬灭(80％甲酰胺，1mg/mL二甲苯C，1mg/mL溴酚蓝，20mM EDTA)，反应产物在95℃变性5分钟，置冰10min。反应混合物在20％的Urea-PAGE分离，再由Typhoon Trio扫描凝胶。
结果如图2所示，持续合成能力可以定义为平均延伸的条带长度，由图2A可知，Dpo4及突变体延伸的主要条带的含量存在明显差异，根据延伸条带荧光值占总延伸的比例，进行加权平均计算DNA聚合酶一次结合DNA延伸的平均长度，并作为衡量持续合成能力的标准可以得到野生型Dpo4的持续合成能力为16nt，与已有的文献数据接近，在已有的文献中当酶比DNA过量20倍时往往能延伸50-100左右的片段，但可以认为是由于不含有Trap而造成的聚合酶重新结合。本实施例中由于Trap的存在，且Dpo4只10倍过量于DNA，因此认为在37℃下，酶10倍过量于DNA时，Dpo4的持续合成能力为16nt。
根据野生型Dpo4的平均长度计算方法计算出所有突变体的平均延伸的条带长度(图2B)，其中Dpo4A181D的延伸长度最长，相比野生型Dpo4，延伸长度增加了25％，Dpo4E63K较Dpo4增加了18.75％，Dpo4V289I、Dpo4A220S、Dpo4M76I、Dpo4F37T的延伸长度相比Dpo4下降，其中Dpo4V289I相比Dpo4下降最为明显下降了18.75％。其余突变体的持续合成能力与野生型Dpo4接近。突变体的持续合成能力结果表明，结合能增加的部分突变体能够增强Dpo4的持续合成能力，结合能减弱的突变体表现出较野生型Dpo4低的持续合成能力。
总的来说，Dpo4E63K、Dpo4N118S、Dpo4A181D、Dpo4I248Y的持续合成能力增加了，而Dpo4F37T、I59M、Dpo4M76I、Dpo4A220S、Dpo4V289I的持续合成能力降低了，Dpo4F33Y、和Sdbh I62V突变对持续合成能力无影响，表明分子动力学模拟手段在一定程度上可以起到预测作用。
实施例4Dpo4和Dpo4突变体的保真性比较
挑选持续合成能力增加明显的Dpo4A181D、Dpo4E63K以及持续能力下降最明显的Dpo4V289I与野生型Dpo4比较在Mn2+条件下的保真性，观察四种模板(表3)在单个碱基(dATP、dCTP、dGTP、dTTP)及混合碱基dNTP条件下的引物延伸情况。20nM的退火好的引物/模板添加到反应缓冲液(10mM HEPES NaOH(pH7.4)，50mM NaCl，10mM MnCl2，200mM dNTPs，1mM DTT，100μg/ml BSA和0.1％的Triton X-100)。加入10nM的DNA聚合酶在37℃起始DNA合成。孵育2h后，利用10μL的终止液淬灭反应，反应产物经20％的Urea-PAGE分离，再由Typhoon Trio扫描凝胶。
结果如图3所示，其中Dpo4及其突变体在四种模板条件下都掺入了正确的核苷酸(图 3A)，但掺入错误核苷酸的情况有明显的差异，Dpo4A181D和Dpo4的错配相比E63K和V289I较为严重，通过错配情况的统计结果来看(图3B)，Dpo4A181D和Dpo4不管是在错配的种类还是错配延伸条带的量均比E63K和V289I明显。Dpo4A181D和Dpo4保真度在Mn2+条件下相差不大，在TA模板下的延伸均为80％左右，TT模板条件下，Dpo4对dTTP的掺入率为88.48％，而Dpo4A181D只有67.47％，但在TG模板下的Dpo4A181D错配较Dpo4严重，Dpo4A181D在此模板下掺入dATP、dTTP、dGTP掺入率分别为73.27％、60.95％、63.07％，而Dpo4的掺入率只有64.94％、59.52％、46.67％。
因此，Dpo4A181D的错误掺入情况比Dpo4严重，保真度相比Dpo4下降，Dpo4E63K只在模板为TT下掺入dTTP达到80％的掺入率，其余错配情况的掺入率均低于60％。Dpo4V289I的错配率在TA模板下掺入dGTP的情况下为67.58％，是Dpo4V289I作用下的最大错配率。
因此由错配的统计结果来看，Dpo4A181D和Dpo4的保真性接近，Dpo4E63K的保真性相比Dpo4和Dpo4A181D有所提高，Dpo4V289I的错配情况最少即保真性最好，保真性由高到低的顺序为：Dpo4V289I、Dpo4E63K、Dpo4、Dpo4A181D，表明持续合成能力的提高与保真性的改变没有必然关系。
表3.保真性实验中所用引物和模板序列
Figure PCTCN2017103118-appb-000003
实施例5Dpo4及突变酶的核苷酸掺入效率比较
比较Dpo4及突变蛋白的核苷酸掺入效率即比较Dpo4及突变蛋白掺入dNTP)的预稳态动力学差异，以120nM的Dpo4及突变蛋白起始30nM(50-mer/15-mer)P/T反应，反应不同时间段后，以EDTA终止聚合反应，产物通过20％聚丙烯酰胺凝胶分离，利用Typhoon定量分析延伸产物的浓度，根据公式[product]＝A(1-exp(-Kobst))计算得到特定dNTP浓度下的反应速率常数，改变dNTP的浓度(20-400μM)，通过SPSS非线性拟合Kobs与dNTP的方程(图 4)Kobs＝Kp[dNTP]/{[dNTP]+Kd,dNTP}计算出核苷酸最大掺入速率Kp和平解离常数Kd,dNTP，从而计算出相应的核苷酸掺入效率(Kp/Kd,dNTP)。
由表4可知，Dpo4A181D和Dpo4E63K，核苷酸掺入速率(Kp)分别是Dpo4的1.8倍和2.6倍，但Dpo4A181D和Dpo4E63K与核苷酸的亲和力(Kd)相比Dpo4降低了1.7和3倍，进而使得三者的核苷酸掺入效率相差不大，Dpo4V289I的核苷酸掺入效率相比于野生型Dpo4下降了45.9％，这种较低的核苷酸掺入效率可能决定了Dpo4V289I相比于Dpo4和其它突变体具有较好的保真性。Kd,dNTP反应了正确核苷酸掺入时基态结合的亲和力，表明了Dpo4与邻近碱基对的相互作用。
根据结果，Dpo4A181D和Dpo4E63K这两个持续合成能力增加的突变体的Kd相比野生型Dpo4，结合的亲和力更弱，可能是突变体增加持续合成能力需要“酶-DNA-dNTP”更快的从“开放状态”转为“关闭状态”，这个过程需要Dpo4的finger和LF区域进行构象变化，能够减弱dNTP与Dpo4之间的作用，使得Dpo4与复制碱基对之间的作用更加松散，从而Kd值增大，同时Dpo4V289I的低持续合成能力对应着较强基态亲和力。
总的来说，除Dpo4V289I的核苷酸掺入效率极大地降低外，Dpo4A181D、Dpo4E63K两个突变体的核苷酸掺入效率与Dpo4基本接近，Dpo4A181D和Dpo4E63K在提高持续合成能力的同时没有影响核苷酸掺入效率。
表4.Dpo4及突变酶的核苷酸掺入效率分析
Figure PCTCN2017103118-appb-000004
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2017103118-appb-000005
Figure PCTCN2017103118-appb-000006
Figure PCTCN2017103118-appb-000007
Figure PCTCN2017103118-appb-000008
Figure PCTCN2017103118-appb-000009
Figure PCTCN2017103118-appb-000010
Figure PCTCN2017103118-appb-000011

Claims (7)

  1. 一种持续合成能力增强的DNA聚合酶Dpo4，其特征在于，将Dpo4第181位的氨基酸A突变为D，或将第63位的E突变为K得到，野生型Dpo4的氨基酸序列如SEQ ID NO.2所示。
  2. 根据权利要求1所述的DNA聚合酶Dpo4，其特征在于，编码野生型Dpo4的核苷酸序列如SEQ ID NO.1所示。
  3. 一种获得权利要求1或2所述持续合成能力增强的DNA聚合酶Dpo4的方法，其特征在于，通过定点突变获得突变体。
  4. 编码权利要求1或2所述持续合成能力增强的DNA聚合酶Dpo4的基因。
  5. 携带权利要求4所述基因的载体或重组细胞。
  6. 含有权利要求1或2所述持续合成能力增强的DNA聚合酶Dpo4的试剂盒。
  7. 权利要求1或2所述持续合成能力增强的DNA聚合酶Dpo4在DNA合成中的应用。
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