第四讲 基因工程的工具酶Enzymes for Gene Engineering 4.1 DNA4.1 DNA工具酶的种类工具酶的种类DNADNA工具酶根据其催化的反应可以分为工具酶根据其催化的反应可以分为5 5大类：大类：（（1 1）核酸酶（）核酸酶（nucleasesnucleases），切开或降解核酸分子切开或降解核酸分子2 2）连接酶）连接酶（（ligasesligases）），将核酸分子连接起来将核酸分子连接起来3 3）聚合酶（）聚合酶（polymerasespolymerases），复制核酸分子的酶复制核酸分子的酶4 4）修饰酶）修饰酶（（modifying enzymemodifying enzyme）），去除或添加，去除或添加 化学基团的酶化学基团的酶5 5）拓扑异构酶）拓扑异构酶（（topoisomerasestopoisomerases）），向共价闭合，向共价闭合环状环状DNADNA分子中引入或消除超螺旋分子中引入或消除超螺旋 Nucleases,any enzyme that cleaves nucleic acids, which belong to the class of enzymes called hydrolases, are usually specific in action, ribonucleases acting only upon ribonucleic acids (RNA) and deoxyribonucleases acting only upon deoxyribonucleic acids (DNA). Processes under control of nucleases are for example protective mechanisms against "foreign" (invading) DNA, degradation of host cell DNA after virus infections, DNA repair, DNA recombination, DNA synthesis, maturation of RNAs or RNA splicing.4.1.1 4.1.1 核酸酶（核酸酶（NucleaseNuclease）） Nucleases are phosphodiesterases with a tremendous variability in their substrate requirements. They are classified by their specificity as EXO- or ENDO-nucleases meaning they require either a free end (exo) to start working or they start from anywhere within a molecule (endo) even when no free ends are available as for example in a covalently closed circle. Some nucleases have both, endo-and exo-activities. Some are specialized for single-stranded DNA (ss-DNA) others for double-stranded DNA (ds-DNA); again, some nucleases can work on both types of DNAs. Further, some exonucleases work in a 3'->5' direction, others in a 5'->3' direction; some do not care and work in both directions. Some endonucleases comprise site-specificity and require distinct sequences for cleavage ,others require helper proteins to find their targets. Single Stranded Nicks in DNAHydrolysis of this ester bondH2OPOOOHO-OH 核酸酶（核酸酶（NucleaseNuclease）） 凡能水解核酸的酶均称为核酸酶凡能水解核酸的酶均称为核酸酶(nuclease)(nuclease)。
核酸核酸酶催化的反应都是使磷酸二酯键水解酶催化的反应都是使磷酸二酯键水解, ,故核酸酶属故核酸酶属磷酸二酯酶磷酸二酯酶 按作用对象可分为按作用对象可分为: :•DeoxyribonucleaseDeoxyribonuclease ( (DNAaseDNAase) ) •RibonucleaseRibonuclease ( (RNAaseRNAase) ) 按水解断裂核酸分子不同方式可分为：按水解断裂核酸分子不同方式可分为： （（1 1））外切核酸酶外切核酸酶( (ExonucleaseExonuclease) )，从，从DNADNA分子分子的末端一个一个地切除核苷酸的末端一个一个地切除核苷酸 （（2 2））内切核酸酶内切核酸酶( (EndonucleaseEndonuclease) )，从，从DNADNA分分子内部打断磷酸二酯键子内部打断磷酸二酯键而内切酶可以在多核苷而内切酶可以在多核苷酸链内的不同位置水解磷酸二酯键酸链内的不同位置水解磷酸二酯键 按水解断裂核酸分子不同方式可分为：按水解断裂核酸分子不同方式可分为： （（1 1））外切核酸酶外切核酸酶( (ExonucleaseExonuclease) )，从，从DNADNA分子分子的末端一个一个地切除核苷酸的末端一个一个地切除核苷酸。
（（2 2））内切核酸酶内切核酸酶( (EndonucleaseEndonuclease) )，从，从DNADNA分分子内部打断磷酸二酯键内切酶可以在多核苷酸子内部打断磷酸二酯键内切酶可以在多核苷酸链内的不同位置水解磷酸二酯键链内的不同位置水解磷酸二酯键 S1S1内切核酸酶内切核酸酶，来源于米曲霉（，来源于米曲霉（AspergillusAspergillus oryzaeoryzae），只切断单链），只切断单链DNADNA或或RNA,RNA,产生带产生带5'5'磷酸的单链磷酸的单链核苷酸或寡核苷酸对双链核苷酸或寡核苷酸对双链DNADNA、双链、双链RNARNA和和DNA-RNADNA-RNA杂杂交体相对不敏感；交体相对不敏感；内切核酸酶内切核酸酶从牛胰腺中制得的从牛胰腺中制得的脱氧核糖核酸酶脱氧核糖核酸酶I I（（DNaseIDNaseI）既能够）既能够切断单链也能够切断双链切断单链也能够切断双链DNaseIDNaseI是一种非特异的内是一种非特异的内切酶，能够打断切酶，能够打断DNADNA内部的任意磷酸二酯键另一方面，内部的任意磷酸二酯键另一方面，称作称作限制性内切酶限制性内切酶的一组特殊的酶能够在特定的位点的一组特殊的酶能够在特定的位点切开双链切开双链DNADNA分子。
分子 DNase I足迹法（DNase I footprinting assay） •蛋白质结合在DNA片段上，能保护结合部位不被DNase破坏，DNA分子经酶切作用后遗留下该片段(亦称“足迹”)，进而可以确定它的序列 RNase RNase H 作用于DNA-RNA杂交分子中的RNA链，用于cDNA文库建立时除去DNA链以便第二条链cDNA链的合成 RNase A 作用于RNA的专一性内切酶，特异 性的作用于嘧啶核苷酸，主要用于除去DNA制备物中的RNA 4.1.2 4.1.2 连接酶（连接酶（DNA ligaseDNA ligase））DNA ligase is a special type of ligase，which is an enzyme that in the cell repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break (a break in both complementary strands of DNA). DNA ligase has applications in both DNA repair and DNA replication. DNA ligases have become an indispensable tool in modern molecular biology research for generating recombinant DNA sequences. For example, DNA ligases are used with restriction enzymes to insert DNA fragments, often genes, into plasmids.The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide with the 5' phosphate end of another. ATP is required for the ligase reaction. DNADNA连接酶：可使一段连接酶：可使一段DNADNA的的3` 3` 羟基末端和羟基末端和5`5`磷酸末端形成磷酸末端形成3`3`，，5`- 5`- 磷酸二酯键，把两个磷酸二酯键，把两个DNADNA片片段连在一起，封闭段连在一起，封闭DNADNA双链上形成的切口的酶。
双链上形成的切口的酶 OH P5`3`3`5`5`3`3`5`DNA ligaseDNA ligase DNA ligase 连接缺刻连接缺刻(nick)(nick) Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required. AATTC· · · · · · GG· · · · · · CTTAAAATTC· · · · · · GG· · · · · · CTTAA5´5´5´5´(1) (2)AATTC· · · · · · GG· · · · · · CTTAAAATTC· · · · · · G G· · · · · · CTTAA5´5´AATTC· · · · · · GG· · · · · · CTTAA5´5´(a)分子间连接(b)分子内连接(1) (2)ATGCTATAGCTAT4连接酶T4连接酶 常用的DNA连接酶有两种：来自大肠杆菌的DNA连接酶（NAD+提供能量）和来自噬菌体的T4DNA连接酶(ATP提供能量）。
二者的作用机理类似 1)DNA1)DNA连接酶不能催化两单链连接酶不能催化两单链DNADNA分子连接；分子连接；2)2)只能连接双链只能连接双链DNADNA分子的单链缺刻分子的单链缺刻(nick)(nick)；；3)3)不能连接双链中一个或多个核苷酸缺失所致不能连接双链中一个或多个核苷酸缺失所致的缺口（的缺口（gapgap）使用时的注意事项 DNA ligase 的活性的活性 4.1.3 4.1.3 聚合酶聚合酶(polymerase)(polymerase)A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA. The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription. In association with a cluster of other enzymes and proteins, they take nucleotides from solvent, and catalyse the synthesis of a polynucleotide sequence against a nucleotide template strand using base-pairing interactions. 聚合酶聚合酶 DNA polymerase A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand. The newly-polymerized molecule is complementary to the template strand. •DNA Polymerase I (or Pol I) is an enzyme that participates in the process of DNA replication in prokaryotes. It is composed of 928 amino acids, and is an example of a processive enzyme - it can sequentially catalyze multiple polymerisations. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase (and, indeed, the first known polymerase of any kind). It was initially characterized in E. coli, although it is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene which encodes Pol I is known as polA. 大肠杆菌大肠杆菌DNADNA聚合酶聚合酶I I（（DNA polymerase I）） 基本用途 大肠杆菌大肠杆菌大肠杆菌大肠杆菌DNADNADNADNA聚合酶聚合酶聚合酶聚合酶I I I I经枯草杆菌蛋白酶处理经枯草杆菌蛋白酶处理经枯草杆菌蛋白酶处理经枯草杆菌蛋白酶处理, , , ,获得获得获得获得N N N N端三端三端三端三分之二的大肽段分之二的大肽段分之二的大肽段分之二的大肽段, , , ,即即即即KlenowKlenowKlenowKlenow酶或酶或酶或酶或Klenow片段片段。
KlenowKlenowKlenowKlenow酶仍拥有酶仍拥有酶仍拥有酶仍拥有5`→3`5`→3`5`→3`5`→3`的的的的DNADNADNADNA聚合酶活性和聚合酶活性和聚合酶活性和聚合酶活性和3`→5`3`→5`3`→5`3`→5`的核的核的核的核酸外切酶活性，但失去了酸外切酶活性，但失去了酸外切酶活性，但失去了酸外切酶活性，但失去了5`→ 3`5`→ 3`5`→ 3`5`→ 3`的核酸外切酶活性的核酸外切酶活性的核酸外切酶活性的核酸外切酶活性KlenowKlenow酶酶酶酶 T4 DNA聚合酶(T4 phage DNA polymerase) The activities of T4 DNA polymerase are very similar to Klenow fragment of DNA polymerase I - it functions as a 5' -> 3' DNA polymerase and a 3' -> 5' exonuclease, but does not have 5' -> 3' exonuclease activity. The 3' -> 5' exonuclease activity of T4 DNA polymerase is roughly 200 times that of Klenow fragment, making it preferred by many investigators for blunting DNAs with 3' overhangs.在无在无dNTPdNTP时，可以从任何时，可以从任何3`-OH3`-OH端外切端外切－在只有一种－在只有一种dNTPdNTP时，外切至互补核苷酸。
时，外切至互补核苷酸 －在四种－在四种dNTPdNTP均存在时，聚合活性占主导地位均存在时，聚合活性占主导地位 •Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965. Taq polymerase•T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. Therefore it replaced the DNA polymerase from E.coli originally used in PCR. Taq's temperature optimum for activity is 75-80°C, with a halflife of 9 minutes at 97.5°C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C. •One of Taq's drawbacks is its relatively low replication fidelity. It lacks a 3' to 5' exonuclease proofreading activity, and has an error rate measured at about 1 in 9,000 nucleotides. Some thermostable DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelity amplification.•Taq makes DNA products that have A (Adenine) overhangs at their 3' ends. This may be useful in , whereby a cloning vector (such as a plasmid) is used which has a T (Thymine) 3' overhang, which complements with the A overhang of the PCR product, thus enabling ligation of the PCR product into the plasmid vector. T7 RNA Polymerase is an RNA polymerase that catalyzes the formation of RNA in the 5'→ 3' direction. T7 polymerase is extremely promoter-specific and only transcribes bacteriophage T7 DNA or DNA cloned downstream of a T7 promoter. The T7 polymerase also requires a DNA template and Mg2+ ion as cofactor for the synthesis of RNA. In contrast to bacterial RNA polymerases, T7 polymerase is not inhibited by the antibiotic rifampicin. The source of the enzyme is the T7 bacteriophage, which is a virus that infects only bacteria. This phage's polymerase has a very low error rate. T7 RNA Polymerase Homogeneously labeled single-stranded RNA can be generated with this system. Transcripts can be non-radioactively labeled to high specific activity with certain labeled nucleotides.T7 polymerase has a molecular weight of 99 kDa. Related family members include phage T3 and SP6 RNA polymerases, but this family is also related to the mitochondrial RNA polymerase.In biotechnology applications, T7 RNA polymerase is commonly used to transcribe DNA which has been cloned into vectors which have two (different) phage promoters (eg, T7 and T3, or T7 and Sp6) in opposite orientation. RNA can be selectively synthesized from either strand of the insert DNA with the different polymerases. 反转录酶反转录酶(reverse transcriptase)Reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into double-stranded DNA. Reverse transcriptase was discovered by Howard Temin at the University of Wisconsin-Madison, and independently by David Baltimore in 1970 at MIT. The two shared the 1975 Nobel Prize in Physiology or Medicine with Renato Dulbecco for their discovery. Well studied reverse transcriptases include: HIV-1 reverse transcriptase from human immunodeficiency virus type 1 M-MLV reverse transcriptase from the Moloney murine leukemia virus AMV reverse transcriptase from the avian myeloblastosis virus 反转录酶具有反转录酶具有反转录酶具有反转录酶具有5 5 5 5′′′′→3→3→3→3′′′′的的的的DNADNADNADNA聚合酶活性，聚合酶活性，聚合酶活性，聚合酶活性，以以以以RNARNARNARNA为模板为模板为模板为模板合成合成合成合成cDNAcDNAcDNAcDNA链链链链, , , ,同时又具有同时又具有同时又具有同时又具有3 3 3 3′′′′ →5→5→5→5′′′′和和和和5 5 5 5′′′′ →3→3→3→3′′′′的的的的RNARNARNARNA外外外外切核酸酶活性切核酸酶活性切核酸酶活性切核酸酶活性。
4.1.4 DNA4.1.4 DNA修饰酶（修饰酶（DNA Modifying Enzymes ）） 通过添加或删除化学基团来修饰通过添加或删除化学基团来修饰DNADNA分子的酶，统分子的酶，统称为称为DNADNA修饰酶碱性磷酸酶碱性磷酸酶多核苷酸激酶多核苷酸激酶 Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment.碱性磷酸酶（碱性磷酸酶（alkaline alkaline phosphatasephosphatase），催化核酸脱），催化核酸脱5`-5`-磷磷酸基团，使酸基团，使DNADNA或或RNARNA片段片段5`-P5`-P末端转换成末端转换成5`-OH5`-OH末端。
末端来源：有两种，分别来自于大肠杆菌和小牛肠道，前者叫来源：有两种，分别来自于大肠杆菌和小牛肠道，前者叫bacterial alkaline bacterial alkaline phosphatasephosphatase (BAP); (BAP); 后者叫后者叫calf calf intestinal alkaline intestinal alkaline phosphatasephosphatase (CIP) (CIP) 碱性磷酸酶的活性碱性磷酸酶的活性 Alkaline phosphatase has become a useful tool in molecular biology laboratories, since DNA normally possesses phosphate groups on the 5' end. Removing these phosphates prevents the DNA from ligating (the 5' end attaching to the 3' end), thereby keeping DNA molecules linear until the next step of the process for which they are being prepared; •One common use in the dairy industry is as a marker of pasteurisation in cows milk. This molecule is denatured by elevated temperatures found during pasteurisation, and can be tested for via colour change of a para-nitro-phenol phosphate substrate in a buffered solution. Raw milk would typically produce a yellow colouration within a couple of minutes, whereas properly pasteurised milk should show no change. •Another important use of alkaline phosphatase is as a label for enzyme immunoassays. （（2 2）多核苷酸激酶）多核苷酸激酶(polynucleotide (polynucleotide kinasekinase) )（从（从T4T4噬菌体侵染的大肠杆菌细胞中提取），和碱性磷噬菌体侵染的大肠杆菌细胞中提取），和碱性磷酸酶的活性相反，在酸酶的活性相反，在DNADNA分子分子5’5’游离末端添加磷酸游离末端添加磷酸基团。
基团Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer of a phosphate from ATP to the 5' end of either DNA or RNA. It is a product of the T4 bacteriophage, and commercial preparations are usually products of the cloned phage gene expressed in E. coli. Polynucleotide kinase 。