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DNA Endonucleases

DNA Endonucleases. • Cleave internal phosphodiester bonds resulting in 3’-OH and 5’-phosphate ends. 5’. 3’-OH. 5’-P. 5’-P. 3’-OH. • some endonucleases cleave randomly (DNase I, II). • Type II Restriction endonucleases are highly sequence specific. EcoRI recognition site :.

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DNA Endonucleases

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  1. DNA Endonucleases • Cleave internal phosphodiester bonds resulting in 3’-OH and 5’-phosphate ends 5’ 3’-OH 5’-P 5’-P 3’-OH • some endonucleases cleave randomly (DNase I, II) • Type II Restriction endonucleases are highly sequence specific EcoRI recognition site: Palindromic site (inverted repeat) • RE are found in bacteria where they are used for protection against foreign DNA

  2. Recognition sequences of some common restriction endonucleases

  3. DNARestrictionEnzyme EcoR V H N O N NH 2 O N N HN N N O N O A•T 5’-GAT ATC-3’ 3’-CTA TAG-5’ Asn185 Thr186

  4. Applications of Restriction Endonucleases in Molecular Biology • DNA fingerprinting (restriction fragment length polymorphism). • 2. Molecular cloning (isolation and amplification of genes).

  5. Southern blotting

  6. Restriction fragment length polymorphisms are used to compare DNA from different sources

  7. DNA Ligase O -O P O DNA Ligase + O- (ATP or NAD+) AMP + PPi O O P O OH O- • Forms phosphodiester bonds between 3’ OH and 5’ phosphate • Requires double-stranded DNA • Activates 5’phosphate to nucleophilic attack by transesterification with activated AMP

  8. DNA Cloning: recombinant DNA technology

  9. Human Genetic Polymorphisms • Human genome size: 3.2 x 109 base pairs • 30,000 genes • 2-4 % of total sequence codes for proteins • Human genetic variation: •  1 sigle nucleotide polymorphism (SNP) per 1,300 bp

  10. Examples of genetic polymorphisms of drug metabolizing enzymes Enzyme substrate examples DNA regions involved cytochrome 2B6 cyclophosphamide exons 1,4,5, and 9 tamoxifen benzodiazepines cytochrome 2D6 debrisoquine internal base changes cytochrome 1A2 caffein 5' flanking region phenacetin N-acetyltransferase aromatic amines

  11. DNA Structure: Take Home Message • Genetic information is stored in DNA. • DNA is a double stranded biopolymer containing repeating units of nitrogen base, deoxyribose sugar, and phosphate. • DNA can be arranged in 3 types of duplexes which contain major and minor grooves. • DNA can adopt several topological forms. • There are enzymes that will cut DNA, ligate DNA, and change the topology of DNA. • Human genome contains about 3.2 billion base pairs. Inter-individual differences are observed at about 1 per 1,000 nucleotides.

  12. Chapter 2: DNA Synthesis (Replication)

  13. DNA Replication is semi-conservative Meselson, Stahl 1958 • Both strands serve as templates for DNA synthesis • Each DNA molecule contains one • strand from original DNA and one • new strand

  14. DNA Polymerization Reaction Requirements for DNA polymerization • Template DNA (single stranded or double-stranded with a “nick”) • A primer strand with a free 3'- hydroxyl group (usually RNA) • Deoxynucleoside 5'-triphosphates (dATP, dGTP, dTTP and dCTP) • Mg2+ to activate the dNTPs • Polymerase and other accessory enzymes General reaction: 2Pi

  15. DNA Polymerization Reaction

  16. DNA Synthesis: addition of new dNTPsfollows Watson-Crick rules O H N N 2 NH N N N N NH 2 O G•C N NH 2 O N N HN N N O A•T Template base Incoming base G C C G T A A T

  17. 3’ 5’ Exonuclease

  18. 5’  3’ -exonuclease

  19. E. coli DNA Polymerase I Klenow Fragment 5' 3' Nucl. Polymerase N 3' 5‘ Nucl. C

  20. Typical Polymerase Structure: E. Coli Pol I fingers thumb palm exonuclease

  21. Polymerase with bound DNA

  22. Mechanism of phosphoryl transfer

  23. Polymerase fidelity mechanisms • Watson-Crick base pairing between the incoming dNTP • and the corresponding base in the template strand. • 2. H-bond formation between the minor groove of the new base pair • and the amino acids in the polymerase active site. • 3. Proofreading mechanism via 3' exonuclease that excises • incorrectly added nucleotides.

  24. 1. Correct Watson-Crick base pairing between the incoming dNTP and the corresponding base in the template strand induces conformational change required for polymerization reaction: Thumb Fingers

  25. 2. H-bond formation between the minor groove of the new base pair and amino acids in the polymerase active site:

  26. All Watson-Crick base pairs contain two H-bond acceptors at the same sites of the minor groove O N H N NH2 2 2 NH N N N N NH NH 2 2 O N NH 2 O N N HN N N HN 2 N O O N A•T N HN N N O G•C O N N NH N N O N T:A C:G

  27. 3. 3’-Exonuclease Proofreading function of DNA polymerases excises incorrectly added nucleotides.

  28. Fidelity of DNA Polymerization: Absolutely Essential!! Error Probability = Polymerization error (10-4) X 3' 5' Nuclease error (10-3) = 10-7 (1 in 10,000,000 nt)

  29. DNA Polymerization Has Three Stages 1) Initiation 2) Priming 3) Processive Synthesis

  30. Problems to overcome: DNA Polymerization • The two strands must be separated, and local DNA over-winding • must be relaxed. The single stranded DNA must be prevented from • re-annealing and protected from degradation by cellular nucleases. 2. Both antiparallel strands must be synthesized simultaneously in the 5’ 3’ direction. 3’ 3. A primer strand is required. 5’

  31. DNA Polymerization: Initiation • • DNA replication begins at a specific site. • •Example: oriC site from E. coli. • • 245 bp out of 4,000,000 bp • • contains a tandem array of three 13-mers; GATCTNTTNTTTT • Synthesis takes place in both directions from the origin (two replication forks)

  32. E. coli replication origin •GATC common motif in oriC •AT bp are common to facilitate duplex unwinding

  33. DNA Polymerization: Initiation • • DNA replication begins at a specific site. • •Example: oriC site from E. coli. • • 245 bp out of 4,000,000 bp • • contains atandem array of three 13-mers; GATCTNTTNTTTT • •GATC common motif in oriC • •AT bp are common to facilitate duplex unwinding • Synthesis takes place in both directions from the origin (two replication forks)

  34. Enzymes involved in the initiation of DNA Polymerization Enzyme Function dnaA recognize replication origin and melts DNA duplexat several sites Helicase (dnaB) unwinding of ds DNA DNA gyrase generates (-) supercoiling SSB stabilize unwound ssDNA Primase (dnaG) an RNA polymerase, generates primers for DNA Pol

  35. Crystal structure of bacterial DNA helicase Stryer Fig. 27.16

  36. DNA helicase: proposed mechanism A1 B1 Stryer Fig. 27.17

  37. Problems to overcome: DNA Polymerization • The two strands must be separated, and local DNA over-winding • must be relaxed. The single stranded DNA must be prevented from • re-annealing and protected from degradation by cellular nucleases. 2. Both antiparallel strands must be synthesized simultaneously in the 5’ 3’ direction. 3’ 3. A primer strand is required. (overall direction) 5’

  38. Lagging strand is synthesized in short fragments (1000-2000 nucleotides long) using multiple primers 3’ 5’

  39. Problems to overcome: DNA Polymerization • The two strands must be separated, and local DNA over-winding • must be relaxed. The single stranded DNA must be prevented from • re-annealing and protected from degradation by cellular nucleases. 2. Both antiparallel strands must be synthesized simultaneously in the 5’ 3’ direction. 3’ 3. A primer strand is required. 5’

  40. A short stretch of RNA is used as a primer for DNA synthesis

  41. Primer Synthesis and removal 3' 5' 5' RNA primer 3' 5' 5' 3' DNA strand 3' 5' 3' 5' DNA Ligase 3' 5' 3' 5'

  42. DNA Ligase seals the nicks O -O P O DNA Ligase + O- (ATP or NAD+) AMP + PPi O O P O OH O- • Forms phosphodiester bonds between 3’ OH and 5’ phosphate • Requires double-stranded DNA • Activates 5’phosphate to nucleophilic attack by trans-esterification with activated AMP

  43. ENZYME O (+)H N 2 Ade P O O(-) 2.E-AMP + P-5’-DNA O AMP-O 5'-DNA P O O O O- 5'-DNA P O AMP-O OH OH O- DNA Ligase -mechanism • E + ATP  E-AMP + PPi O 3. DNA-3' OH DNA-3' 5'-DNA + O P O O- + AMP-OH

  44. What is the function of RNA priming? • DNA polymerase tests the correctness of the preceding base pair • before forming a new phosphodiester bond • de novo synthesis does not allow proofreading of the first • nucleotide • Low fidelity RNA primer is later replaced with DNA

  45. DNA Synthesis Helicase Gyrase SSB Primase 3' 5' DNA Pol III DNA Pol I 5' DNA Ligase 5' 3' 3' 5'

  46. E. coli Pol III: an asymmetrical dimer with two Polymerase sites Polymerase Polymerase Stryer Fig. 27.30

  47. Lagging strand loops to enable the simultaneous replication of both DNA strands by dimeric DNA Pol III Stryer Fig. 27.33

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