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Chapter 2

Chapter 2. Enzymes for Genetic Engineering. 2.1 Restriction endonucleases 2.2 DNA ligase 2.3 DNA polymerase 2.4 Other Enzymes 2.5 probe labeling techniques. 第二章 基因工程的工具酶 教学目的、要求： 1. 了解基因工程各种工具酶的基本概念、分类及在基因工程中的作用； 2 ．掌握限制性内切酶，聚合酶，连接酶，反转录酶，修饰酶的生物学特性和应用； 教学内容： 1 、工具酶与基因工程 2 、限制酶

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Chapter 2

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  1. Chapter 2 Enzymes for Genetic Engineering 2.1 Restriction endonucleases 2.2 DNA ligase 2.3 DNA polymerase 2.4 Other Enzymes 2.5 probe labeling techniques

  2. 第二章 基因工程的工具酶 教学目的、要求： 1. 了解基因工程各种工具酶的基本概念、分类及在基因工程中的作用； 2．掌握限制性内切酶，聚合酶，连接酶，反转录酶，修饰酶的生物学特性和应用； 教学内容： 1、工具酶与基因工程 2、限制酶 3、DNA聚合酶 4、DNA连接酶 5、S1核酸酶 6、BAL31核酸酶 7、碱性磷酸酶 8、逆转录酶 9、T7和SP6RNA聚合酶

  3. Enzymes for DNA analysis and molecular cloning

  4. 2.1 Restriction endonucleases 2.1.1 Restriction modification system (R-M ) 2.1.2 nomenclature 2.1.3 three types of RE 2.1.4 Factors Affecting Restriction Enzyme Digestion

  5. 2.1 Restriction endonucleases (限制性核酸内切酶） • Restriction Enzymes are: - Proteins - Cleave DNA inside with a sequence-specific manner - Different restriction enzymes in different organisms - Evolved as a defense mechanism against infection by foreign viruses - Isolated from bacteria，algae and archaea (古生菌 )

  6. Facts: • the first restriction enzyme, HindII, was isolated in 1970 by Hamilton Smith and his colleagues[1] • the 1978 Nobel Prize for Physiology or Medicine was awarded to Daniel Nathans, Werner Arber, and Hamilton Smith. • Over 3000 restriction enzymes have been studied in detail • More than 600 of these are available commercially • Routinely used for DNA modification and manipulation in • laboratories. Roberts RJ (April 2005). "How restriction enzymes became the workhorses of molecular biology". Proc. Natl. Acad. Sci. U.S.A.102 (17): 5905–8. Roberts RJ, Vincze T, Posfai J, Macelis D. (2007). "REBASE--enzymes and genes for DNA restriction and modification". Nucleic Acids Res35 (Database issue): D269–70.

  7. 2.1.1 Restriction modification system (限制与修饰系统） Restriction Enzymes have evolved to provide a defense mechanism against invading viruses. [1] Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction. Host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. Collectively, these two processes form the restriction modification system (R-M system). Arber W, Linn S (1969). "DNA modification and restriction". Annu. Rev. Biochem.38: 467–500. Kobayashi I (September 2001). "Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution". Nucleic Acids Res. 29 (18): 3742–56

  8. Bacteria phage‘s invation causes host cells dead and lead to form plaques Lawn 菌膜 Plaque 嗜菌斑

  9. W. Arber and S. Linn (1969) E. coli C • Plating efficiencies of bacteriophage λ (l phage) grown on E. coli strains C, K-12 and B: Phageλ E.Coli C plaque （＋） E.Coli K-12 plaque （－） E.Coli B plaque （－） • E.coliK-12 and B can resist phage λ. • The DNA of phage which had been grown on strains K-12 and B were found to have chemically modified bases which were methylated.

  10. Additional studies with other strains indicate that different strains had specific methylated bases. EcoRI vs. EcoRIMethylase R-M for this strain 5’—GAAmTTC—3’ will not be cut by EcoRI 3’—CTTAmAG—5’

  11. In addition to possessing a particular methylase, individual bacterial strains also contained accompanying specific endonuclease activities. A characteristic feature of the sites of methylation, was that they involved palindromic（回文）DNA sequences.EcoR1 methylase specificity（Rubin and Modrich, 1977） The REs cleaved at or near the methylation recognition site. However, they would not cleave at these specific palindromic sequences if the DNA was methylated.

  12. Methylation occurres at very specific sites in the DNA • Typical sites of methylation include theN6 position of adenine, the N4 position, or the C5 position of cytosine. Assignment1: what are the methylating sites for Guanine and Thymine?

  13. 2.1.2 Nomenclature (命名） Since their discovery in the 1970s, many different restriction enzymes have been identified in different bacteria. Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus（属）, species（种） and strain（菌株）. eg. EcoRI restriction enzyme was derived as follow

  14. Each restriction enzyme has specific recognition sequence Enzymes with 6-bp Recognition sequence Bgl II “bagel-two” Bacillus globigi 5’-AGATCT-3’ 3’-TCTAGA-5’ Pst I “ P-S-T-one” Providencia stuartii 5’-CTGCAG-3’ 3’-GACGTC-5’ Enzyme with4-bp Recognition sequence Hha I “ha-ha-one” Haemphilus haemolyticus 5’-GCGC-3’ 3’-CGCG-5’ Sau3A “sow-three-A” Staphylococcus aureus 3A 5’-GATC-3’ 3’-CTAG-5’ Enzyme with 8-bp Recognition sequence Not I “not-one” Nocardia 5’-GCGGCCGC-3’ otitidis-caviarum 3’-CGCCGGCG-5’

  15. Restriction endonucleases are categorized into three general groups (Types I, II and III) based on • composition (protein subunits) • enzyme cofactor requirements (Mg2+, SAM, ATP) • the nature of their target sequence • the position of their DNA cleavage site relative to thetarget sequence. 2.1.3 Types of Restriction Enzymes

  16. Type I restriction enzymes • Possess three subunits called HsdR, HsdM, and HsdS: • HsdR is required for restriction • HsdM is necessary for adding methyl groups to host DNA • HsdS is important for specificity of cut site recognition in • addition to its methyl transferase activity. • multifunctional enzymes, multimers • Enzyme cofactors required for their activity: • S- Adenosyl methionine (AdoMet or SAM) • hydrolyzed adenosine triphosphate (ATP) • magnesiumions (Mg2+ ) Me doner Energy doner Cleavage activity activator

  17. Target sequence: The recognition site is asymmetrical and is composed of two portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a spacer of about 6–8 nucleotides. eg. EcoB: 5’-TGANNNNNNNNTGCT-3’ Cleavage site: cut at a site that differs, and is some distance (at least 1000 bp) away, from their recognition site. Recognition site ≠ Cutting site

  18. Type III restriction enzymes Contain more than one subunit Double functions (cut and methylase) Require Mg2+, AdoMet and ATP cofactors for their roles in DNA methylation and restriction, respectively Recognize two separate non-palindromic sequences that are inversely oriented （反向排列）. e.g. EcoP1: AGACC EcoP15 : CAGCAG Cut DNA about 20-30 base pairs out side the recognition site with unpredictable manner.

  19. Type II restriction enzymes • Dimers (consists of two identical subunits) • Usually require only Mg2+ as a cofactor for their • enzyme activity • Restriction activity but no modification • Recognition sites are usually undivided and • palindromic and 4–8 nucleotides in length • Each cuts in a predictable manner, at a site • within or adjacent to the recognition sequence • The most commonly available and used restriction enzymes

  20. Structure of EcoRI dimer

  21. In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily nomenclature was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes. These subgroups are defined using a letter suffix. Type IIB restriction enzymes (e.g. BcgI and BplI) are multimers, containing more than one subunit. They cleave DNA on both sides of their recognition to cut out the recognition site. They require both AdoMet and Mg2+ cofactors. Type IIE restriction endonucleases (e.g. NaeI) cleave DNA following interaction with two copies of their recognition sequence. One recognition site acts as the target for cleavage, while the other acts as an allosteric effector that speeds up or improves the efficiency of enzyme cleavage. Similar to type IIE enzymes, type IIF restriction endonucleases (e.g. NgoMIV) interact with two copies of their recognition sequence but cleave both sequences at the same time.Type IIG restriction endonucleases (Eco57I) do have a single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active. Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylated DNA.Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites. These enzymes may function as dimers. Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits. Some recognize palindromic sequences while others have asymmetric recognition sites

  22. Commonly used RE, Recognition Sites

  23. Occurring frequencies of recognition sites E.Coli: genome DNA 4.2 x 106 bp, EcoRI frequency: 4.2 x 106 x 1/46 = 4.2 (cut sites in whole genome DNA)

  24. GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGT Eco RI GGCCTGCG AATTCCCGATCGAAGGCCCG AATTCTGGCCA CCGGACGCTTAA GGGCTAGCTTCCGGGCTTAA GACCGGT Examples 2 cuts with 3 fragments. GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGT Hae III GG CCTGCGAATTCCCGATCGAAGG CCCGAATTCTGG CCA CC GGACGCTTAAGGGCTAGCTTCC GGGCTTAAGACC GGT 3 cuts with 4 fragments.

  25. Two major ways for RE cleave Symmetrical cutting • “blunt ends”

  26. “Sticky ends”-I Asymmetrical cutting 3’OH of the sticky end is very important for re-joining of DNA fragments. 5’-P: required for phosphodiester bond formation 5’- overhanging sticky ends

  27. “Sticky ends”-II Pst1 Providencia stuartii 3’overhanging sticky end

  28. 2.1.4 Factors Affecting Restriction Enzyme Digestion • DNA Purity • Cross contamination • Metylation: dam(+) dcm(+) strains GATC  GAmTC; CCWGG  CCmWGG BamHI  GGATCC ? • Temperature and time • Buffer: will give the optimum pH, ionic strength, Mg2+, • Star activities: a relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimum for the enzyme. • Reaction volume and RE amount W=Purine base

  29. 2.2 DNA ligase DNA ligase is a special type of ligase, which is basically an enzyme that in the cell repairs single-stranded discontinuities in double stranded DNA molecules ( strands that have double-strand break, a break in both complementary strands of DNA). Purified DNA ligase is used in gene cloning to join DNA molecules together. The alternative, a single-strand break, is fixed by a different type of DNA ligase using the complementary strand as a template but still requires DNA ligase to create the final phosphodiester bond to fully repair the DNA. Okazaki fragment（冈崎片段？）

  30. Ligase mechanism 3’ 5’ 5’ 5’ 5’ 3’ nick: lack P-diester bound gap: nucleotide missing 缺刻 缺口 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.

  31. Energy-dependent joining of the chains Activated by NAD+ or ATP hydrolysis E. coli DNA lygase: NAD  NMN+ + AMP T4 DNA lygase: ATP  AMP + PPi AMP -attaches to lysine group on enzyme AMP transferred to 5’ phosphate at ligation site 3’ OH at ligation site splits out AMP and joins to 5’ phosphate Ligation needs:

  32. NH2-Lysine- NH2-Lysine- + High Energy Nitrogen Phosphate Bond N H 2 N N NMN+ O N N O - P - C H O 2 O H O O H Ligase Mechanism NAD+ Activated Phosphorylating complex AMP-Lys-ligase complex

  33. NH2-Lysine- OH O O P O O N H 2 N N O N N O - P - C H O 2 O H O O H

  34. NH2-Lysine- OH O O O O P O O O O N H 2 N N O N N O - P - C H O 2 O P H O O H + AMP

  35. The formation of phosphodiester bound ATP is required for the ligase reaction.

  36. One vital, and often tricky, aspect to performing successful recombination experiments involving the ligation of cohesive-ended fragments is controlling the optimal temperature. Most experiments use T4 DNA Ligase (isolated from bacteriophage T4), which is most active at 25°C. However, in order to perform successful ligations with cohesive-ended fragments, the optimal enzyme temperature needs to be balanced with the melting temperature Tm (also the annealing temperature) of the DNA fragments being ligated. In general, 14-16 °C, over night (o/n) Thinking: Why the temperature is the critical factor affecting ligation?

  37. If the ambient（周围）temperature exceeds Tm, homologous pairing of the sticky ends will not occur because the high temperature disrupts hydrogen bonding. Since blunt-ended DNA fragments have no cohesive ends to anneal, controlling the optimal temperature becomes much less important. The most efficient ligation temperature will be the temperature at which T4 DNA ligase functions optimally. Therefore, the majority of blunt-ended ligations are carried out at 20-25°C.

  38. Joining of stick ends and blunt ends Use of linkers:

  39. Use of adaptors:when the restriction enzyme can also cut the DNA fragment, using an adaptor to join DNA fragments may considered. an adaptor containing sticky end with 5’-OH modified terminus to avoid self ligation.

  40. Produce sticky ends by homopolyer tailling: use of terminal deoxylnucleotidyl transferase Add a series of poly nucleotides onto the 3’-terminal of a dsDNA. Only need to add one dNTP into the test tube when conduct the polymerase catalyzing reaction.

  41. Topoisomerase mediated TA cloning: Topoisomerase is both a restriction enzyme and ligase naturally involving in DNA replication. • Topoisomerase I from vaccinia virus binds dsDNA at specific sequence and cleave it after the 5’-CCCTT in one strand. • The energy from the broken bond is conserved by formation of a covalent bond between the 3’- phosphate of the cleavaged DNA strand and the 274 tyrosine residue of topoisomerase I. • The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5’ hydroxyl of the original cleaved strand , reversing the reaction and releasing topoisomerase. (Shuman, 1991-1994)

  42. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single A to the 3’-ends of PCR products. Linearized vector DNA with overhanging 3’-T, and covalently bound to the topoisomerase I PCR products inserts to ligate efficiently with the vector. RT: 5 min, Vol: 6 ul

  43. PCR products inserts to ligate efficiently with the vector.

  44. topoisomerase, originally termed gyrase, was first discovered by Taiwanese Harvard Professor James C. Wang.[ The double-helical configuration that DNA strands naturally reside in makes them difficult to separate, and yet they must be separated by helicase proteins if other enzymes are to transcribe the sequences that encode proteins, or if chromosomes are to be replicated. In so-called circular DNA, in which double helical DNA is bent around and joined in a circle, the two strands are topologically linked, or knotted. Otherwise, identical loops of DNA having different numbers of twists are topoisomers, and cannot be interconverted by any process that does not involve the breaking of DNA strands. Topoisomerases catalyze and guide the unknotting of DNA by creating transient breaks in the DNA using a conserved Tyrosine as the catalytic residue. Type I topoisomerase cuts one strand of a DNA double helix, relaxation occurs, and then the cut strand is reannealed. • Type II topoisomerase cuts both strands of one DNA double helix, passes another unbroken DNA helix through it, and then reanneals the cut strand. It is also split into two subclasses: type IIA and type IIB topoisomerases, which share similar structure and mechanisms. Examples of type IIA topoisomerases include eukaryotic topo II, E. coli gyrase, and E. coli topo IV. Examples of type IIB topoisomerase include topo VI.

  45. 2.3 DNA polymerase DNA polymerase: an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand alone the template 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 and identical to the template's original partner strand. DNA polymerases use a magnesium ion for catalytic activity.

  46. 2.3.1 DNA polymerase I and Klenow fragment Structure: single peptide with different domains Functions: 5’3' Polymerase activity 3’5' exonuclease (proofreading) 5’3' exonuclease activity (RNA Primer remove)

  47. Reaction condition for noncell system: 4 dNTPs (substrates) Mg2+ Peimers with 3’-OH (no known DNA polimerase is able to begin a new chain) DNA template: template reading 3’ 5’ new chain extension 5’3’

  48. The Klenow fragment is a large protein fragment produced when DNA pol I from E. coli is enzymatically cleaved by the protease subtilisin.

  49. Because the 5' → 3' exonuclease activity of DNA pol I makes it unsuitable for many applications, the Klenow fragment, which lacks this activity, can be very useful in research. The Klenow fragment is extremely useful for research-based tasks such as: • Synthesis of double-stranded DNA from ssDNA • templates • Filling in (meaning removal of overhangs to create • blunt ends) recessed 3' ends of DNA fragments • Digesting away protruding（凸出的） 3' overhangs • Preparation of radioactiveDNA probes

  50. 2.3.2 T4 and T7 DNA polymerase Source: Bacterophageies of E.coli Template dependent ＤNApolymerase T4 DNA polymerase Function：Similar to Klenow fagment 3’-5’ cleavage (exonuclease) Characteristics：In general, T4 DNA polymerase is used for the same types of reactions as Klenow fragment, particularly in blunting the ends of DNA with 5' or 3' overhangs.

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