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Prokaryotic and eukaryotic dna replication with their clinical applications

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A comprehensive presentation on Prokaryotic and Eukaryotic DNA Replication with their clinical applications for MBBS , BDS, B Pharm & Biotechnology students to facilitate self- study.

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Prokaryotic and eukaryotic dna replication with their clinical applications

  1. 1. Prokaryotic and Eukaryotic DNA Replication with their clinical applications Dr. Rohini C Sane
  2. 2. Identical base sequences 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ WatsonandCrickdouble-strandedDNAstructureTwisted Ladder (B form and double helix) 1. Right handed helix (two deoxy ribonucleotide strands around each other ) 2. Anti- parallel strands : (5’→3’ &3’→5’ ) 3. Width (diameter ): 20A (2 nm ) 4. Each turn of pitch = 34 nm (3.4nm )with 10 base pairs at the distant of 3.4nm 5. Hydrophobic bases stacked inside & hydrophilic deoxyribose nucleotides periphery 6. Stands are identical but complementary due to base pairing
  3. 3. Hydrogen bonds holding two strands of DNA Structure Watson & Crick Model of DNA Structure :Two stands are held together by hydrogen bonds, Hydrogen bonds between Purine & Pyrimidine ,A= T ,C  G (CG Pair stronger than at pair)
  4. 4. Major and minor grooves in DNA structure Each turn of pitch = 34 nm (3.4nm )with 10 base pairs at the distant of 3.4nm
  5. 5. DNA as a long term repository of genetic information 1. DNA is a bank of genetic information & controls protein biosynthesis. 2. Single mammalian cell contains 10 picograms (10-12 gm )of DNA. 3. DNA (long term repository of genetic information) is more stable than RNA. 4. DNA in cell must be duplicated ,maintained & accurately pass down to the daughter cell.
  6. 6. Functions of Deoxy-ribonucleic Acids(DNA) ❖Functions of Deoxy-ribonucleic Acids(DNA): 1. Chemical basis of hereditary & reserve bank of genetic information 2. Maintain identity of different species over million years 3. Control cellular functions 4. DNA possess organized genes which control protein synthesis 5. DNA→RNA→PROTEIN ( central dogma) 6. Mitochondrial DNA → specified function with respect to protein synthesis
  7. 7. Organization of Prokaryotic and Eukaryotic DNA • Organization of Prokaryotic DNA: as a single chromosome in in the form of double-stranded circle. It is packed in the form of nucleotides by interaction with proteins and certain cations( polyamines ). • Organization of Eukaryotic DNA: DNA is associated with Histone proteins to form chromatin which then gets organized into compact structures namely chromosomes. • Nucleosomes : two turns of DNA(150bp)wrapped around core(Histone proteins H1,H2B,H3,H4 two molecules each) .
  8. 8. Packing of DNA into chromosomes: 1. Level 1 Winding of DNA around histones to create a nucleosome structure. 2. Level 2 Nucleosomes connected by strands of linker DNA like beads on a string. 3. Level 3 Packaging of nucleosomes into 30-nm chromatin fiber. 4. Level 4 Formation of looped domains. Organization of Eukaryotic DNA 30 nm fibers organized into loops by anchoring the fiber at A/T rich regions (scaffold –associated regions SARS ) to protein scaffold. During mitosis, the loops are further coiled , the chromosomes condense and become visible. 
  9. 9. The central dogma of molecular biology:1 ❑The central dogma of molecular biology states that genetic information flows DNA to RNA to Protein by processes of 1. DNA Replication :that involves formation of daughter DNA molecules using a parental DNA as a template . 2. Transcription of DNA: in which genetic information in DNA is transcribed to form messenger RNA(m- RNA). 3. Translation of m-RNA into amino acid sequence of protein. ❖DNA Replication →Transcription of DNA →Synthesis of m-RNA →Translation of m-RNA →Protein Synthesis ❖In retroviruses which have RNA genomes, genetic information flows in reverse direction i.e. from RNA to DNA.
  10. 10. The central dogma of molecular biology DNA RNA Protein Transcription Translation Reverse Transcription Replication The flow of genetic information
  11. 11. The central dogma of molecular biology:2
  12. 12. Basic requirements for DNA replication ➢The basic requirements and components of replication are the same for prokaryotes and eukaryotes. ➢Replication in prokaryotes is much better understood than in eukaryotes. • Substrates: 4 deoxyribonucleosides triphosphate( dATP , dGTP ,dCTP, dTTP) • Template : separated strands of DNA serve as template for the synthesis of the new daughter DNA strands .It is required to direct the addition of the appropriate complementary nucleotide to newly synthesized DNA strand. • Proteins and Enzymes : different types of DNA polymerases and proteins
  13. 13. ❖DNA Replication involves three R : 1. Replication 2. Repair 3. Recombination
  14. 14. Proteins involved in initiation of DNA replication at E.Coli origin Protein Molecular weight Numberof subunits Function DNA A Protein 52000 1 Recognizes ori sequences, opens DNA duplex at a specific site in origin DNA B Protein(Helicase) 300000 6 Unwinds DNA , primosome constituent DNA C Protein 29000 1 Required for DNA B binding at origin HU(Histone like protein) 19000 2 DNA binding protein , stimulates initiation Primase(DNA G Protein) 60000 1 SynthesizesRNAprimers,primosomeconstituent SSB(singlestrandedbinding protein) 75600 4 BindssinglestrandedDNAandstabilizestheseparated DNAandpreventsrenaturationofDNA DNAtopoisomeraseII (DNAgyrase) 400000 4 releasestorsionalstraingeneratedbyDNAunwinding DNA polymerase I 103000 1 Filling gaps and excisions of primers DNA polymerase III 791500 17 new DNA strand elongation DNA ligase 74000 1 Sealsthesinglestrandnickbetweenthenascentchain andokazaki fragmentsonlaggingstrand(ligation)
  15. 15. Proteins involved in initiation of DNA replication at E.Coli origin Protein Molecular weight Numberof subunits Function RNA polymerase 454000 5 Facilitates DNA A Protein binding activity DNA methylase 32000 1 Methylate of (5’ ) GATC sequence at ori C DNA polymerase I Filling gaps and excisions of primers Ter binding protein Prevents DNA B Protein(Helicase) from further unwinding of DNA and facilitates the termination of replication
  16. 16. Comparison of Prokaryotic and Eukaryotic DNA polymerases Prokaryotic DNA polymerases Eukaryotic DNA polymerases Functions I (Pol)  Gap filling and synthesis between Okazaki fragments of lagging strand(5’ →3’ polymerization activity) II(Poll)  DNA proof reading and DNA repair(3’→5’exonuclease activity)  DNA repair (5’→3’ exonuclease activity)  Mitochondrial DNA synthesis III(Polll)  Functions at replication fork, catalyzing Leading and lagging strand synthesis
  17. 17. DNA Replication in Prokaryotes ❖When cell divides a daughter cell receives identical copies of genetic information from a parent cell. ❖Definition of DNA Replication :Replication of DNA is the process in which DNA copies produce identical daughter molecules of DNA. 1. DNA Replication exhibits high fidelity which is essential for survival of fetus. 2. DNA Replication is semi- conservative :half of original DNA is conserved in the daughter DNA .(Meselson & Stahl 1958) • Newly synthesized DNA has half of the parental DNA & one half of new DNA.
  18. 18. Mechanism of DNA Replication In Prokaryotes ❖Features of DNA Replication in Prokaryotes: • Semi- continuous, semi–conservative & bi-directional • Replication proceeds in 5’→3’ direction • Simultaneously both strands of DNA • Replication in Leading strand is continuous & forward . • Replication in Lagging strand is discontinuous & short pieces of DNA (15-250 nucleotides ).Okazaki fragments are produced on Lagging strand . • DNA Synthesis :bidirectional from point of origin in replication bubble • Two replication forks move in opposite directions from replication bubble or replication eye ,which becomes lager and assumes a  shaped structure. • 3 Stages of replication : initiation ,elongation and termination.
  19. 19. DNA Replication in Prokaryotes: produce identical daughter molecules of DNA DNA replication : Duplication /synthesis of DNA. It is needed to transfer the genetic information stored in DNA of parent cell to a daughter cell during cell reproduction. In semi conservative mechanism , each replicated duplex daughter DNA molecules contains one parent strand and one newly synthesized strand.
  20. 20. DNA Replication in Prokaryotes: Semiconservative : half of original DNA is conserved in the daughter DNA .
  21. 21. Three models for DNA replication
  22. 22. Components of DNA Replication
  23. 23. Components of DNA Replication • Replicon : is the unit of DNA in which individual acts of replication occurs. Bacterial chromosome contains a single replicon ,eukaryotic chromosome has a large number of replicons. • Replication fork: also known as growing point ,at which replication occurs. Replication may be unidirectional or multidirectional based on whether one or more replication forks starts from the origin respectively . • Origin of Replication : the site at which replication begins .These sites are generally AT – rich to facilitate unwinding . Proteins and enzymes required assembled at origins.
  24. 24. Replication forks of DNA in prokaryotes and eukaryotes
  25. 25. Overview of bacterial DNA replication Twotopologicallyinterlinkedcircular chromosomescalledcatenanes generatedbyreplicationare separatedbyatypeII topoisomeraseandsegregatedinto twodaughtercellsatcelldivision.
  26. 26. Directionality of the DNA strands at a replication fork Leading strand Lagging strand Fork movement A new strand of DNA is always synthesized in the 5==3 direction, with the free 3 OH as the point at which the DNA is elongated Replication fork
  27. 27. Direction of movement of replication fork
  28. 28. DNA replication in prokaryotes :Semi- continuous & bi-directional DNA Replication in Leading strand is continuous & forward. Replication in Lagging strand is discontinuous & short pieces of DNA (15-250 nucleotides). Okazaki fragments are produced on Lagging strand .
  29. 29. Semi–conservative DNA Replication In Prokaryotes
  30. 30. Replication proceeds in 5’→3’ direction DNA Synthesis : bidirectional from point of origin in replication bubble
  31. 31. Functions of Helicases in DNA Replication ❑Helicases separate double stranded DNA to single stranded DNA during replication and the energy derived from ATP hydrolysis . ❑Helicases unwind the DNA duplex just ahead of the replication fork at a rate of 1000 bp/s. Two DNA Helicases unwind DNA at a replication fork moving in opposite directions ,one on the leading strand template and another on the lagging strand template. ❖Functions of Helicases: 1. DNA unwinding occurs during replication. Helicases in conjunction with topoisomerase relieve torsional stain . 2. It functions in homologous recombination, nucleotide excision repair , transcription termination and conjugation.
  32. 32. DNA helicase unwinds the DNA duplex ahead of DNA polymerase creating single stranded DNA that can be used as a template Functions of Helicases in DNA Replication
  33. 33. Functions of Helicases in DNA Replication in Prokaryotes: DNA unwinding during replication. Helicases in conjunction with topoisomerase relieve torsional stain . Role of Helicases in DNA Replication
  34. 34. Uses energy from ATP to unwind the duplex DNA SSB SSB SSB SSB Functions of Helicases in DNA Replication
  35. 35. Functions of single binding proteins(SSB) in DNA Replication ❖Single binding proteins(SSB):also known as DNA helix destabilizing proteins or Single stranded DNA binding proteins. They have no enzyme activity. ❖Functions of SSB Proteins during DNA Replication: 1. Keep two strands of DNA separate(separated by helicases). 2. Bind tightly in a co-operative manner to single stranded DNA (separated strands) and makes it available as a template for DNA Replication/ synthesis. 3. Stabilize DNA in a single strand state and prevent base pairing. 4. Protect single stranded DNA degradation by nucleases.
  36. 36. Role of single binding proteins(SSB) in DNA Replication:1 ssDNA binding proteins bind to the sugar phosphate backbone leaving the bases exposed for DNA polymerase.
  37. 37. Role of single binding proteins(SSB) in DNA Replication:2 ssDNA binding proteins are required to “iron out” the unwound DNA Stabilize DNA in a single strand state and prevent base pairing. Protect single stranded DNA degradation by nucleases.
  38. 38. Role of  clamp in Elongation of DNA Replication in Prokaryotes ❖ As the leading strand is being synthesized ,corresponding portion of Lagging strand is looped through a  clamp enabling coordinate synthesis of both strands. ❖Both core complex and  clamp dissociate after synthesis of Okazaki fragments and again associate the next Okazaki fragment .
  39. 39. DNA polymerase is not very processive (i.e. it falls off the DNA easily). A “sliding clamp” is required to keep DNA polymerase on and allow duplication of long stretches of DNA Role of  clamp in Elongation of DNA Replication in Prokaryotes
  40. 40. Role of sliding clamp  -subunits in Elongation of DNA Replication in Prokaryotes Astheleadingstrandisbeingsynthesized,correspondingportionofLaggingstrandisloopedthrougha clampenablingcoordinatesynthesisofbothstrands.Bothcorecomplexandclampdissociateaftersynthesis ofOkazakifragmentsandagainassociatethenextOkazakifragment.
  41. 41. A “clamp loader:” complex is required to get the clamp onto the DNA. Role of clamp loader
  42. 42. Role of Helicases , SSB proteins and Topoisomerase in DNA Replication Topoisomerase type I and type II play important role in DNA Replication , transcription and recombination.
  43. 43. Positive and Negative supercoils of DNA PositivesupercoilsofDNAareformedwhenthe DNAmoleculeistwistedinthesamedirectionasthe righthandedhelixofB-formDNAaboutitsaxis. NegativesupercoilsofDNAareformedwhentheDNA moleculeistwistedintheoppositedirectionasthe righthandedhelixofB-formDNAaboutitsaxis.
  44. 44. Activities of enzymes topoisomerase type I / II and supercoils of DNA Positive supercoils of DNA Negative supercoils of DNA( are formed when the DNA molecule is twisted in the same direction as the right handed helix of B-form DNA about its axis. are formed when the DNA molecule is twisted in the opposite direction as the right handed helix of B-form DNA about its axis. Introduced by topoisomerase I and relaxed by topoisomerase II. Introduced by topoisomerase II and relaxed by topoisomerase I. The amount /activities of enzymes topoisomerase type I and II are regulated to maintain appropriate degree of negative supercoiling.
  45. 45. Supercoils and DNA Topoisomerase • Super coils are formed as double helix separates from one side & replication proceed at the other side (twisted ropes pooled apart) • DNA Topoisomerase Type I –nuclease activity –cuts single strand (to overcome problem of supercoiling) & reseal the strand by ligase activity. • DNA Topoisomerase Type II(called DNA Gyrase in prokaryotes): cuts both strands (to overcome problem of supercoiling) & reseal the strands by Ligase activity. It introduces negative supercoils to DNA using free energy from ATP hydrolysis. ❑Cancer treatment ❖Camphotherin –an inhibitor of DNA Topoisomerase Type I ❖Amasacrime & Etoposide- inhibitors of DNA Topoisomerase TYPE II
  46. 46. Functions and Mechanism of action of Topoisomerase in DNA Replication:1 Topoisomerase • Relax torsional strain generated during DNA unwinding by helicases by causing a transient break in DNA followed by resealing . Topoisomerases bind covalently to DNA and cleave a phosphodiester bond that is reformed after the enzyme dissociates from the DNA. • Removes knots and catalyze catenation(linking together of double stranded, circular DNA) and decatenation. • Type I Topoisomerase cleave only one strand and catenate/decatenate substrate containing a nick ,whereas Type II Topoisomerase enzyme cleaves both strands of DNA and catenate/decatenate covalently closed cycle.
  47. 47. Functions and Mechanism of action of Topoisomerase in DNA Replication:2
  48. 48. Functions of Helicases and Topoisomerase in DNA Replication in Prokaryotes Helicases in conjunction with topoisomerase relieve torsional stain .
  49. 49. Able to covalently link together Unable to covalently link the 2 individual nucleotides together 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ DNA Polymerase Cannot Initiate new StrandsRole of RNA Primer of DNA Replication In Prokaryotes:1 ❖ Theprimer: ▪ isrequiredforDNA synthesis ▪ ShortpieceofRNA(10 nucleotides) ▪ synthesizedbyDNA dependentRNA Polymerase (alsocalled primase)in5’to3’ direction)usingDNAas template ▪ DNAPolymeraseinitially addsadeoxynucleotide to3’OHgroupofthe primerandthen continuestoadd deoxynucleotidesto3’ endofthegrowing strand.
  50. 50. RNA primer is synthesized by RNA polymerase/primase along with single stranded binding proteins and forms primosomes . Function of RNA Primase of DNA Replication In Prokaryotes
  51. 51. DNA primase synthesizes an RNA primer to initiate DNA synthesis on the lagging strand Synthesis of RNA Primer by DNA Primase PrimerissynthesizedbyDNA dependentRNAPolymerase (also calledprimase)in5’to3’direction) usingDNAastemplate DNAPolymeraseinitiallyadds adeoxynucleotideto3’OHgroup of theprimerandthencontinues toadddeoxynucleotidesto3’end ofthegrowingstrand.
  52. 52. Role of RNA Primer of DNA Replication In Prokaryotes:2 ❖RNA Primer of DNA Replication In Prokaryotes: • RNA Primer or initiator RNA ( iRNA ) –A short fragment of RNA 4-12 nucleotides in length base paired to the template DNA and provides free 3’OH group for initiating DNA synthesis by DNA polymerase . • RNA primer is synthesized by RNA polymerase along with single stranded binding proteins and forms primosome . • Leading stand needs a single primer. • Lagging stand need constant synthesis & supply of RNA primers . • Extension of RNA Primer is carried out by DNA polymerase III which functions as a dimer with one core complex of holoenzyme moving continuously along the Leading stand template while other cycles from one Okazaki fragment to the other on the Lagging stand . • RNA Primer is removed by DNA pol I and subsequent gap filling is done by the same enzyme.
  53. 53. Synthesis and replacement of RNA primers during DNA replication
  54. 54. DNA polymerases ❖DNA –dependent DNA polymerase catalyze DNA synthesis on DNA template during replication, following DNA damage ,recombination, after removal of the primer from the lagging strand.
  55. 55. DNA polymerases in E. coli
  56. 56. Types of E. coli DNA polymerases DNA polymerase I It is also known as Kornberg enzyme. It contains 1 or 2 atoms of Zn++ at active site. DNA polymerase II It is minor component during growth. It is induced by SOS response. DNA polymerase III It is the most active DNA polymerase. It has the highest rate of chain elongation and Processivity among E. coli DNA polymerases. There are five Types of E. coli DNA polymerases of which polymerase I,II and III studied extensively .
  57. 57. Genes coding E coli DNA polymerases DNA polymerase I A single polypeptide encoded by the pol A gene DNA polymerase II encoded by the pol B gene DNA polymerase III encoded by pol C, dna E, dna N, dna q genes
  58. 58. Enzyme activities of E coli DNA polymerases DNA polymerase I 5’ →3’ polymerase has both 5’ →3’ and 3’ → 5’exonuclease activities proteolytic cleavage yields a large C terminal Kienow fragment with polymerase and 3’ → 5’exonuclease exonuclease activity and a small fragment with 5’ →3’ exonuclease activities DNA polymerase II 5’ →3’ polymerase has 5’ →3’ ’exonuclease activity and lacks 3’ → 5’exonuclease activity DNA polymerase III 5’ →3’ polymerase has 3’→5’exonuclease activity
  59. 59. Functions of E coli DNA polymerases DNA polymerase I Gap filling and exonuclease activity ding replication , repair and recombination Nick translation DNA polymerase II proofreading DNA repair DNA polymerase III Enzyme functions in form of a large complex called DNA polymerase III with 13 subunits.  four subunits that function as a sliding clamp (increase Processivity) . The  complex functions as a clamp loader.
  60. 60. Comparison of E coli DNA polymerases:1
  61. 61. Comparison of E coli DNA polymerases:2
  62. 62. Comparison of Eukaryotic DNA polymerases
  63. 63. DNA polymerases III Of E.coli
  64. 64. 5’ →3’ polymerization activity of DNA polymerase • DNA –dependent DNA polymerase catalyze DNA synthesis on DNA template by successive addition of dNTPs to the free 3’ OH of growing DNA chain by following reactions : • (dNMP)n + dNTP→ (dNMP)n+1 +PPi • The mechanism involves nucleophilic attack of free 3’ OH group on the phosphorous atom of incoming dNTP with release of PPi and formation of diester bonds. PPi is hydrolyzed by pyrophosphatase pulling the reaction to completion in vivo. ❖The enzyme requires : 1. A primer containing a free 3’ OH group base paired to the template 2. All four dNTPs 3. Mg2+ ions ➢The enzyme has low processivity and cannot synthesis long DNA.
  65. 65. Hydrolysis of DNA by nucleases 1. Those enzymes which hydrolyze only from the end of DNA molecule are called exonucleases. 2. Those enzymes which hydrolyze the internal phosphodiester bonds of DNA molecule are called endonucleases. 3. certain endonucleases cut at a specific sequence of DNA ; these molecular scissors are called restriction endonucleases (RE).
  66. 66. 5’→3’ exonuclease activity of DNA polymerase ❖This essential for 1. Removal of primer 2. Repair of damaged DNA 3. Excision of nonpaired stench of DNA o a segment containing chemically modified or mutated nucleotides
  67. 67. 3’→5’exonuclease activity of DNA polymerase ❖It serves proofreading function and ensures high fidelity of DNA replication ( only 1 error for ever 10 9 nucleotides or 1 wrong nucleotide /10000 cells) ❖proofreading function removes : 1. A primer terminus not base paired with the template 2. Incorrect nucleotides added to the growing chain 3. Frayed end of a primer cased by partial melting
  68. 68. Processivity of DNA polymerase • Processivity refers to the number of nucleotides added to the nascent chain before the polymerase dissociate from the template. • Processivity of DNA polymerase can be increased by an accessory protein functioning as a sliding clamp that holds the polymerase firmly while it moves on the DNA and release the enzyme at a region on the double stranded DNA . Assembly of the clamp requires a clamp loader.
  69. 69. Proof Reading Function of DNA Polymerase III • Fidelity is maintained by proof reading function of DNA Polymerase III. • Checks the incoming nucleotide & allows only complementary base (matched base ) to be added to growing strand. • Edits its mistakes –if any and removes wrongly added /placed nucleotide.
  70. 70. Schematic representation of DNA Polymerase III Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA
  71. 71. Stages of DNA replication ❖The process of replication is divided into 3 stages : 1. Initiation 2. Elongation 3. Termination
  72. 72. Initiation of DNA Replication in Prokaryotes
  73. 73. Initiation of DNA Replication in Prokaryotes • Prokaryotic cells lack nuclei and have single circular double stranded chromosomal DNA . Circular DNA duplex unwinds in such a way as to form an eye or bubble (replication bubble). • Initiation of DNA Replication involves unwinding (separation) of two complementary strands and formation of replication fork. • Unwinding occurs at a single site with specific DNA sequence on a circular DNA . This site is called the origin of replication (ori). • Replication bubble provides two points at which replication occurs (active synthesis)to form replication fork. • Replication of double stranded DNA is bidirectional i.e. replication forks move in both directions away from the origin. • One round of replication involves synthesis of over 4 million nucleotides in each new strand and completed within 40 minutes. • Replication ends at termination point on the other side of DNA .
  74. 74. Importance of Replication Bubbles during DNA Replication In Prokaryotes Bubble is formed –as two complementary strands of DNA separate . Multiple replication bubbles are formed –essential to enhance replication process .
  75. 75. Initiation site (ori)of DNA Replication in Prokaryotes ❖Initiation of DNA Replication in Prokaryotes: 1. DNA Replication in Prokaryotes is initiated at a oriC. (a single site of origin of replication) 2. Initiation site is a short DNA sequence of 245 base pairs containing four 9 base pair sequences (nanomers) and three A-T rich 13 base pair sequences (13 –mers) . 3. Many molecules of DNA A (a specific protein) bind to nanomers of a oriC (an initiation site ) in a cooperative manner with melting of 13 –mers. This causes separation of two strands of DNA . 4. In Eukaryotes ,there are multiple sites of origin of replication of DNA. These multiple sites almost exclusively composed of A-T base pairs along the DNA helix and referred as a Consensus sequence.
  76. 76. Initiation site(ori)of DNA Replication in Prokaryotes ❖Two series of short repeats at initiation site of DNA replication in Prokaryotes: 1. three repeats of a 13 bp sequence and 2. four repeats of a 9 bp sequence
  77. 77. DNA sequences at the Bacterial origin of Replication Three A-T rich 13 base pair sequences (13 –mers) initiation site of DNA replication
  78. 78. Steps involved in initiation of DNA Replication in Prokaryotes • DNA A protein recognizes and binds to the ori of the DNA and successively denatures the DNA . • Loading of DNA B(helicase) by DNA C to unwound region of DNA is activated by DNA A . Release of DNA C from DNA B-DNA C complex after loading. • DNA Helicases: binds to both replication fork & moves along DNA helix separating two strands .It is a Zip opener and dependent on ATP for energy supply. • Unwinding of DNA occurs bidirectionally by DNA B creating two replication forks.(Separation of two strands of parent DNA results in formation of replication fork-Site of active DNA synthesis). • The replication fork moves along using the parent DNA as a template and the daughter DNA molecule synthesized. • Torsional stress induced by unwinding relieved by DNA gyrase/Topoisomerase by cutting either one or both DNA strands . • SSB Stabilizes two separated DNA strands and prevent their reassociation.
  79. 79. Role of DNA A proteins in Replication at oriC • DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences – causes the region to wrap around the DnaA proteins and separates the AT-rich region Loading of DNA B(a helicase) by DNA C to unwound region of DNA activated by DNA A . Release of DNA C from DNA B-DNA C complex after loading. Unwinding of DNA occurs bidirectionally by DNA B creating two replication forks. Separation of two strands of parent DNA results in formation of replication fork(a site of active DNA synthesis)
  80. 80. Formation and functions of primosome • OncetheDNAstrandsareseparated,thebindingofprimase(DNAGprotein)takesplace. • Primaseformsacomplexwithproteinsknownasprimosome.Primosomeisformedat eachofreplicatingforks. • Functionofprimosome:primosomerecognizeaspecificsiteonDNAwhereRNAprimer synthesisoccurs.
  81. 81. Topoisomerase Protein complexes of the replication fork DNA polymerase DNA primase DNA Helicase ssDNA binding protein Sliding Clamp Clamp Loader DNA Ligase DNA Topoisomerase
  82. 82. “Three Dimensional” view of Replication Fork Direction of fork movement Direction of synthesis Of lagging strand Direction of synthesis of leading strand
  83. 83. Elongation of DNA Replication in Prokaryotes
  84. 84. Elongation of DNA Replication in Prokaryotes • Once each primer has been laid down ,two DNA polymerase III complexes assembled( one at each of the prime sites). • Because of antiparallel nature of the two strands , the synthesis of DNA along the two strands is different.
  85. 85. The polarity of DNA synthesis creates an asymmetry between the leading strand and the lagging strand at the replication fork. Differential synthesis of DNA along the two strands
  86. 86. Elongation of DNA Replication in Prokaryotes ❖ Elongation of DNA Replication in Prokaryotes: • Two new strands are synthesized in simultaneously in 5’→3’direction by DNA pol III and in opposite directions . The DNA synthesis can proceed only in 5’→3’direction. • One strand which runs in the 3’→ 5’ direction .It is replicated continuously by DNA pol III in the 5’→3’ direction towards replication fork is the Leading strand. This strand requires only one primer. • Other strand which runs in the 5’→ 3’ direction .This strand is replicated discontinuously by DNA pol III in the 5’→3’ direction(in the direction opposite to the movement of replication fork) . The short fragments of 1 -2 base pairs are termed as Okazaki fragments. This new strand is known as the Lagging stand . It needs numerous RNA primers at specified intervals for synthesis of segments of DNA. • Thus, RNA Priming is required for synthesis of both Leading stand and Lagging stand .
  87. 87. Elongation of DNA Replication in Prokaryotes
  88. 88. Two dimensional view of a replication fork Direction of synthesis on leading strand 3’ 5’ 3’ 5’ 3’ 5’ Synthesis of two new strands in simultaneously in opposite direction by DNA Polymerase III Leading strand : in a direction 5’→3’ towards replication fork &continuous Lagging strand: a direction 5’→3’ away from replication fork & Discontinuous Synthesis of Leading strand
  89. 89. Synthesis of Okazaki fragments in DNA replication of prokaryotes
  90. 90. Replication of the Lagging Strand
  91. 91. Synthesis of Okazaki fragments in lagging strand during DNA replication of prokaryotes In Synthesis of Okazaki fragments in DNA replication of prokaryotes • Incoming Deoxy ribonucleotides are added one after other and to 3’ end of growing DNA chain. • Pyrophosphate (Ppi) is removed by addition of each nucleotide. • Template strand (parent strand)determines the base sequence of newly synthesized complementary DNA.
  92. 92. A 3’ hydroxyl group is necessary for addition of nucleotides ElongationofDNAstrandduringreplication:IncomingDeoxyribonucleotidesareaddedoneafterotherand to3’endofgrowingDNAchain.Pyrophosphate(Ppi)isremovedbyadditionofeachnucleotide.
  93. 93. Lagging strand synthesis
  94. 94. NeedofOkazakifragmentsofLaggingstrandduringDNAReplicationinProkaryotes • Need of Okazaki fragments of DNA Replication in Prokaryotes is due to Polarity problem. • Leading strand : its 3’ end (3’OH) oriented towards the fork therefore elongation by sequential addition of new nucleotides. • Lagging strand : no DNA polymerase to add nucleotides to 5’ end of growing chain (3’→5’ direction ) • In Lagging strand DNA synthesis occurs as series of small fragments called Okazaki pieces/fragments in normal 5’→3’ direction& later on Okazaki fragments ligated to form continuous strand. • Okazaki fragments ligation is done by DNA ligase & DNA polymerase I.
  95. 95. Action of DNA Polymerase I • Upon the completion of lagging and lagging strand synthesis ,the RNA primers are removed from fragments by DNA polymerase I . • This polymerase fills the gaps that are produced by removal of the primer . • But it cannot join two polynucleotide chains together hence additional enzyme DNA ligase is needed.
  96. 96. Replacement of RNA Primer by DNA Polymerase I • RNA primer is excised by DNA Polymerase I &takes its position. • DNA Polymerase I catalyzes DNA synthesis in 5’ →3’ direction replaces RNA primer • DNA Ligase –catalyzes formation of phospho-diester linkage between DNA synthesized by DNA Polymerase III and small fragment of DNA produced by DNA Polymerase I. • Sealing –requires energy –(ATP→ AMP +Ppi) • DNA Polymerase II –DNA repair process
  97. 97. Synthesis and replacement of RNA primers during DNA replication
  98. 98. Functions/ Action of DNA ligase ❖Function of DNA ligase: 1. Repairs single strand nicks in duplex DNA 2. the joining of ends of okazaki fragments 3. Links the ends of linear double – stranded DNA to form circles. ➢This enzyme catalyzes the formation of phosphodiester bond between the 3’ OH group at the end of one DNA chain and 5’ phosphate group ate end of other. ➢This energy needed for this process is supplied by NAD+ in bacteria and ATP in eukaryotes.
  99. 99. Mechanism of action of ligase in DNA Replication DNA ligase catalyzes the formation of a phosphodiester bond between two DNA fragments. The cofactor for the enzyme is NAD+ in E.coli and ATP in Eukaryotes. Reaction mechanism : The enzyme reacts with NAD+ (ATP) to from a covalent adenylyl enzyme intermediate (E-AMP) and nicotinamide mononucleotide (NMN) PPi. E+ NAD+ (ATP)  E –AMP + NMN ( PPi ) The adenylyl group is transferred from E –AMP to 5’phosphate of DNA to form a pyrophosphate bond . Nucleophilic attack of 5’phosphate by 3’OH group of the adjacent nucleotide forms a phosphodiester bond.
  100. 100. Function of DNA polymerase I and ligase in sealing of okazaki fragments
  101. 101. Nick translation • Nick translation involves degradation of a DNA (RNA) segment by 5’ →3’ ’exonuclease activity of DNA polymerase I , followed by Gap filling polymerase activity and nick sealing by ligase .This is useful in radiolabeling nucleic acid strand.
  102. 102. Nicks are single strand breaks in double stranded DNA DNA ligase seals nicks left by lagging strand replication Nick translation
  103. 103. Proofreading Function of DNA Polymerases ❖Fidelity( accuracy) of DNA replication is maintained by proofreading function of DNA Polymerase III. ❖DNA Polymerase III: 1. Checks the incoming nucleotide & allows only complementary base (matched base ) to be added to growing strand. 2. Edits its mistakes –if any and removes wrongly added /placed nucleotide. ➢Pol I and Pol II are known to excise nucleotides before the introduction of the next nucleotide (Proofreading activity). ➢Incorrect nucleotide are incorporated with a frequency 1 in 108 -1012 bases, which could lead to mutation .The error ratio during replication is kept at a very low level. ➢Mismatches (the incorrect interactions) occur more frequently but do not lead to stable incorporations because of all three DNA polymerase have 3’ to 5’ exonuclease activity (Proofreading activity) .
  104. 104. DNA Polymerase III and DNA Replication of Prokaryotes ❖DNA Polymerase III • DNA Polymerase III catalyzes DNA synthesis • Substrate for DNA Polymerase III (Prerequisite for replication) –is four types of Deoxyribonucleotide triphosphate (dATP ,dGTP, dCTP, dTTP) • DNA synthesis occurs in 5’ → 3’ Direction ( Anti-parallel to the parent template strand ) ❑Synthesis of two new strands in simultaneously in opposite direction by DNA Polymerase III a) One in a direction 5’→3’ towards replication fork &continuous b) Other in a direction 5’→3’ away from replication fork & Discontinuous
  105. 105. Template strand (parent strand)determines the base sequence of newly synthesized complementary DNA.
  106. 106. Termination of DNA Replication in Prokaryotes
  107. 107. Termination of DNA Replication in Prokaryotes ❖Termination of DNA Replication in Prokaryotes: • Two replication forks meet at a specific sequence(termination sequence) of 2 base pairs called Terminator region that bind to a protein factor called Tus (for Terminus Utilization Substance). • A specific protein ,Ter binding protein binds to these sequences (Terminator region) and prevent helicase (DNA B protein) from further unwinding of DNA . This facilitates the termination of replication. • Tus- Ter complex arrest replication fork in one direction and other fork halts when it encounters the arrested replication fork. • Two topologically interlinked circular chromosomes called catenanes generated by replication are separated by a type II topoisomerase and segregated into two daughter cells at cell division.
  108. 108. Role of Tus-Ter in Termination of DNA Replication in Prokaryotes Tus-Tercomplexarrestreplicationforkinonedirection andotherforkhaltswhenitencountersthearrested replicationfork.
  109. 109. Tus-Ter complex arrest replication fork in one direction Two replication forks meet at a specific sequence of 2 base pairs called Terminator region (Ter) that bind to a protein factor called Tus (for Terminus Utilization Substance).
  110. 110. Overview of termination of bacterial DNA replication Twotopologicallyinterlinkedcircular chromosomescalledcatenanes generatedbyreplicationare separatedbyatypeII topoisomeraseandsegregatedinto twodaughtercellsatcelldivision.
  111. 111. Summary of Functions of enzymes /proteins of DNA synthesis in prokaryotes:1
  112. 112. SummaryofFunctionsofenzymes/proteinsofDNAsynthesisinprokaryotes:2
  113. 113. Types and functions of Eukaryotic DNA polymerases Five types of DNA polymerases in Eukaryotes : Pol , Pol , Pol , Pol  and Pol  ❖Pol  : 1. present in the nucleus. 2. has polymerase activity ,lack proofreading function. 3. is involved in the synthesis of short primers that are extended by Pol  . 4. provides the template for replication factor C (RFC) ,an accessory factor that loads DNA polymerases (similar to E.coli Pol  complex) .Pol  loaded by RFC binds to the initiation complex at the origin and synthesizes an RNA primer of 1 base pairs followed by 2 -3 base pairs of DNA called initiator DNA (iDNA).The iDNA is then attached by polymerase /. This is known as pol switch. ❖Pol  : 1. present in the nucleus as a dimer. 2. has low lowest fidelity and processivity. 3. functions in DNA repair. ❖Pol  :is involved in mitochondrial replication . ❖Pol  : is involved in leading strand synthesis and functions in DNA repair. ❖Pol  : is the main enzyme in DNA replication .
  114. 114. Functions of Eukaryotic DNA polymerases Pol  ❖Pol  : 1. is the main enzyme in DNA replication . 2. synthesizes both leading and lagging strands . 3. has proofreading function. 4. binds proliferating cell nuclear antigen (PCNA) , an accessory factor that functions as a sliding clamp and increases processivity.(similar to  subunit of E.coli Pol III)
  115. 115. Comparison of Prokaryotic and Eukaryotic DNA polymerases Prokaryotic DNA polymerases Eukaryotic DNA polymerases Functions I (Pol l)  Gap filling and synthesis between Okazaki fragments of lagging strand(5’ →3’ polymerization activity) II(Pol ll)  DNA proof reading and DNA repair(3’→5’exonuclease activity)  DNA repair (5’→3’ exonuclease activity)  Mitochondrial DNA synthesis III(Pol III)  Functions at replication fork, catalyzing Leading and lagging strand synthesis
  116. 116. Eukaryotic DNA polymerases : Pol ,Poland Pol  Polbindsproliferatingcellnuclearantigen (PCNA),anaccessoryfactorthatfunctionsas aslidingclampandincreasesprocessivity Polisinvolvedinleadingstrand synthesisandfunctionsinDNArepair. Pol  is involved in the synthesis of short primers that are extended by Pol .
  117. 117. Eukaryotic DNA Replication • In Eukaryotic DNA Replication occurs in the S phase of the cell cycle. • Eukaryotic DNA Replication is bidirectional occurring at the multiple sites simultaneously . • The Replication origins are present in clusters called Replication units. In human ,there are about 1 ori of replication consisting of 1 base pairs each. • Each replicon consist of replication bubbles with two replication forks moving in opposite directions. Replication continues until the replication bubbles merge together. • The mechanism is similar to that seen in prokaryotes. • There are 5 different types of DNA polymerases which catalyze replication and repair . (Pol , Pol , Pol , Pol , Pol  )
  118. 118. S phase of the cell cycle
  119. 119. Prokaryotic and Eukaryotic DNA Replication In Eukaryotic DNA Replication occurs in the S phase of the cell cycle. Eukaryotic DNA Replication is bidirectional occurring at the multiple sites simultaneously .
  120. 120. Origins in Eukaryotic DNA Replication The Replication origins of Eukaryotic DNA Replication are present in clusters called Replication units. In human ,there are about 1 ORI of replication consisting of 1 base pairs each. Each replicon consist of replication bubbles with two replication forks moving in opposite directions. Replication continues until
  121. 121. origins in Eukaryotic DNA Replication Inyeastcells,theDNAsequenceknownasanautonomouslyreplicatingsequence(ARS)composedalmost exclusivelyofA-Tbasepairs.ARSisthesitefortheoriginofreplicationcomplex(ORC).
  122. 122. Originofreplicationcomplex(ORC)ineukaryoticreplication ❖Originofreplicationcomplex(ORC):madeofsixproteins. ▪ Replicationcomplex:(ORC)sixproteins+licensingfactors ▪ Functionoflicensingfactors:permitstheformationoftheinitiatingcomplex.These proteinsservetoensurethateachrepliconisreplicatedonceandonlyonceinacellcycle. ▪ Destructionoflicensingfactors:aftertheformationofinitiationcomplex ▪ ProcessofDestructionoflicensingfactors:Destruction ismarkedwhenitistaggedby Ubiquitinpresentinacell(presentinalleukaryoticcells).Thisisfollowedby proteasomaldigestionoftheubiquitintaggedproteins. ▪ DNAhelicase: separatestheparentDNAstrandsandarestabilizedbybindingof replicationproteinA(singlestrandedDNAbindingprotein). ▪ Polymerase : the initiator DNA polymerization(DNA polymerase activity) and synthesizes RNA primers(primase activity). It lacks exonuclease activity. ▪ Function of protein replication factor C(RFC):displaces of Polymerase  and attracts proliferating cell nuclear antigen (PCNA).
  123. 123. Polymerase switching • Functions of proliferating cell nuclear antigen (PCNA): PCNA binds to DNA polymerase  ( function similar to polymerase III of E.Coli). The binding of PCNA to polymerase  , increases enzyme processivity and starts replicating long stretches of deoxyribonucleotides . • This process is called polymerase switching because polymerase  replaces polymerase .
  124. 124. Functions of Polymerase  ❖Functions of Polymerase : 1. DNA polymerization(replicating long stretches of deoxyribonucleotides) . 2. 3’→ 5’ exonuclease activity : edits/repairs the replicated DNA 3. Replication by Polymerase  continues in both directions from the origin of replication until adjacent replicons meet and fuse . ➢RNA primers are removed by RNase H and the DNA fragments are ligated by DNA ligase .
  125. 125. Functions of RNase H1 and FEN1 during Eukaryotic DNA Replication • The okazaki fragments in mammals are removed by RNase H1 ,which makes an endonucleolytic cut , and FEN1 that cleaves primer . • The newly synthesized DNA is packaged into nucleosomes by proteins termed as chromatin assembly factors .
  126. 126. Replication forks in Eukaryotic DNA Replication The mechanism of Eukaryotic DNA replication is similar to that seen in prokaryotes.
  127. 127. Removal of the okazaki fragments in mammals by RNase H1 (an endonucleolytic cut) and cleavage of RNA primer by FEN1
  128. 128. Sliding clamp of the beta subunit Pol  which synthesizes both leading and lagging strand binds PCNA ,a cyclin that functions as a sliding clamp.
  129. 129. Chromosomal DNA is packaged and organized at several levels. Each phase of condensation or compaction and organization decreases overall DNA accessibility to an extent that the DNA sequences in metaphase chromosomes are almost totally transcriptionally inert. In toto, these five levels of DNA compaction result in nearly a 104-fold linear decrease in end-to-end DNA length. Complete condensation and decondensation of the linear DNA in chromosomes occur in the space of hours during the normal replicative cell cycle The newly synthesized DNA is packaged into nucleosomes by proteins termed as chromatin assembly factors .
  130. 130. End replication problem in Eukaryotic DNA Replication
  131. 131. End replication problem in Eukaryotic DNA Replication:1 • In Eukaryotes replicating the ends of the lagging strand becomes a problem because there is no template available for the RNA primer of the last Okazaki fragments to bind. • As a result ,there is an overhang the telomeres with gradual shortening of the chromosomes after each round of replication. • Telomeres shortening is considering to be responsible for aging. • Telomerase, an RNA- protein enzyme complex that carries an RNA template ,recognizes the G – rich tip of a telomere sequence and extends it in 5’→3’ direction before replication. • Consequently ,the 3’ end of the telomere is longer than the 5’ end and forms a T loop which protects against nuclease attack.
  132. 132. End replication problem in Eukaryotic DNA Replication:2 ❖Extension of the parental strands by telomerase is followed by: 1. Strand separation 2. Synthesis of new strands 3. Removal of primers 4. Gap filling The newly synthesized strands that are not shorter than the parental strands.
  133. 133. End replication problem in Eukaryotic DNA Replication:3 Extensionoftheparentalstrandsbytelomeraseisfollowedbystrandseparation,synthesisofnewstrands, removalofprimersandGapfilling Thenewlysynthesizedstrandsthatarenotshorterthantheparentalstrands.
  134. 134. End replication problem in Eukaryotic DNA Replication:4 Afterthenormalreplication,thereisasinglestrandinthisregion,sotheportionisdegradedbyexonucleases. Thisbrokenendleadstoaberrantrecombinationorendtoendfusions.Unlessthereissomemechanismto replicatetelomeres,thelengthofthechromosomewillgoonreducingateachcelldivision.Thestabilityofthe chromosomesisthuslost.Manygenesmightbelostintheprocess.
  135. 135. Telomeres
  136. 136. Telomeres Replication always takes place from 5’to 3’ direction in the new strand. The DNA polymerase enzyme is not able to synthesize the new strand at the end of 5’ end of the new strand . In other words ,a small portion of ( about 300 nucleotides couldn't be replicated).
  137. 137. Importance of Telomeres • Replication always takes place from 5’to 3’ direction in the new strand. The DNA polymerase enzyme is not able to synthesize the new strand at the end of 5’ end of the new strand . In other words ,a small portion of ( about 300 nucleotides couldn't be replicated). • This end piece of chromosome is called as Telomeres. Therefore enzyme Telomerase or Telomere Terminal transferase takes up the job of replication of the end piece of chromosomes . The Telomeres are noncoding repetitive sequences . • After the normal replication ,there is a single strand in this region, so the portion is degraded by exonucleases. This broken end leads to aberrant recombination or end to end fusions . • Unless there is some mechanism to replicate telomeres ,the length of the chromosome will go on reducing at each cell division .The stability of the chromosomes is thus lost. Many genes might be lost in the process. • The shortening of Telomeres end is prevented by an enzyme Telomerase. It contains an RNA component ,which provides the template for telomeric repeat synthesis.
  138. 138. Telomeres and aging Terminalrestrictionfragmentsfrom70yearsoldindividualsareshorterthanthosefrom20yearsold individuals.Thusinoldage,theTelomeraseactivityislost,leadingtochromosomeinstabilityandcelldeath. Telomerasemayberesponsiblefortheimmortalizationofcancercells.
  139. 139. Characteristics of Telomerase Telomerase : ▪ is present in microorganisms, plants , animals and germ –line cells of human . ▪ It acts as a reverse transcriptase .The Telomerase recognizes 3’ end of telomeres and then a small DNA strand is synthesized. ▪ Terminal restriction fragments from 70 years old individuals are shorter than those from 20 years old individuals .Thus in old age ,the Telomerase activity is lost, leading to chromosome instability and cell death. ▪ is absent in normal human somatic cells but present in the cancer cells. ▪ may be responsible for the immortalization of cancer cells. ▪ is a potential target for anticancer agents .
  140. 140. Role of Telomerase The end piece of chromosome is called as Telomeres. Therefore enzyme Telomerase or Telomere Terminal transferase takes up the job of replication of the end piece of chromosomes . The Telomeres are noncoding repetitive sequences .
  141. 141. Telomerase acts as a reverse transcriptase Telomerase acts as a reverse transcriptase .The Telomerase recognizes 3’ end of telomeres and then a small DNA strand is synthesized.
  142. 142. Telomerase and human diseases Telomerase is also implicated in other human diseases which include : 1. Cancer 2. Diabetes mellitus 3. Aplastic anemia 4. Fanconi’s anemia 5. Bloom syndrome 6. Ataxia telangiectasia
  143. 143. Telomerase and cancer cells As a general rule, cancer cells have continued presence of Telomerase and the chromosome length is maintained ,leading to continued cell division .As the cancer cells have increased and persistent activity of Telomerase , the cancer cells become immortal .
  144. 144. Telomerase and cancer cells The shortening of Telomeres end is prevented by an enzyme Telomerase. It contains an RNA component ,which provides the template for telomeric repeat synthesis.
  145. 145. The use of antisense oligonucleotides against RNA component of the Telomerase arrests the uncontrolled cell proliferation with the minimum side effects. Mechanismofantisense oligonucleotideagainst RNA componentto preventCancercell proliferation
  146. 146. Telomerase and cancer cells Telomeraseisabsentinnormalhumansomaticcellsbutpresentinthecancercells.Itmayberesponsiblefor theimmortalizationofcancercells.Itisapotentialtargetforanticanceragents.
  147. 147. Telomerase and chemotherapy ➢Telomerase is a therapeutic target for cancer cell chemotherapy . ➢ Inhibition of Telomerase can effectively control the multiplication of malignant cells . ➢The use of antisense oligonucleotides against RNA component of the Telomerase arrests the uncontrolled cell proliferation with the minimum side effects.
  148. 148. Telomerase and chemotherapy
  149. 149. Telomerase is a target for cancer therapy
  150. 150. Comparison of Prokaryotic and Eukaryotic DNA Replication Features Prokaryotic DNA Replication Eukaryotic DNA Replication Numberofreplicationorigins Single Multiple Rate of replication 500 to 1000 nucleotides/sec (10 times faster than in eukaryotes) 50-100 nucleotides/sec Types of DNA polymerases 5(Pol designated by Roman numerals I,II,III,IV,V) 14(Pol designated by Greek numerals ,,,, etc.) RNA primer length 50 nucleotides 9 nucleotides RNA primer removal DNA pol I RNase H Nucleotide length Okazaki fragments of lagging strand 1000 – 2000 nucleotides 200 nucleotides Strand elongation DNA pol III DNA pol , Telomerase absent Present Sliding clamp Sliding clamp PCNA
  151. 151. Types of DNA damage ❖Types of DNA damage include : ➢Single and double stranded beaks ➢Cross linking between • Bases in the same strand • Bases in the opposite strands • DNA and protein ➢Bases changes such as : • Insertions • Deletions • Covalent modification ➢ substitutions which may be: • Transitions (replacement of one purine/pyrimidine by another purine/pyrimidine) • Transversions (replacement of purine by another a pyrimidine or vice versa) ➢Oxidative damage by reactive oxygen species.
  152. 152. Causes of DNA damage ❖Causes of DNA damage may be 1. Spontaneous 2. Induced
  153. 153. Spontaneous DNA damage ❖Spontaneous DNA damage include : 1. Deamination of cytosine to uracil that base pairs with A instead of G 2. Depurination (apurination) 3. Depyrimidination
  154. 154. Spontaneous DNA damage Deamination of cytosine to uracil that base pairs with A instead of G
  155. 155. Induced DNA damage ❖DNA damage induced by : 1. Radiation 2. Chemicals ➢DNA damage induced by Radiation: 1. Ionizing radiation (X -rays and  -rays) which causes depurination ( apurination) and hydrolysis of phosphodiester bonds. 2. UV radiation from sunlight forms thymine- thymine dimer between successive T residues, leading to replication errors .
  156. 156. Induced DNA damage by radiations UVradiationfromsunlightformsthymine-thymine dimerbetweensuccessiveTresidues,leadingto replicationerrors. Induced DNA damage by Ionizing radiation (X -rays and  -rays) on Guanine
  157. 157. Induced DNA damage
  158. 158. DNA damage induced by Chemicals ❑DNA damage induced by Chemicals 1. Alkylating agents that are strong electrophiles that bind to DNA. Examples include methylmethane sulphonate (MMS) and ethyl nitrosourea. 2. Non-Alkylating agents such as nitrous acids ,which causes deamination of cytosine ,Adenine and guanine to uracil, hypoxanthine and xanthine respectively. 3. Intercalators containing aromatic rings that insert between adjacent base pairs in DNA e.g. Daunorubicin produced by strains of Streptomyces(anticancer agent). 4. Base analogs such as bromodeoxyuridine (BroU) which is analog of T which is incorporated into DNA instead of T. It mispairs with G instead of A, resulting in AT to GC transition.
  159. 159. DNA damage induced by Non-Alkylating agents Non-Alkylating agents such as nitrous acids ,which causes deamination of cytosine ,Adenine and guanine to uracil, hypoxanthine and xanthine respectively .
  160. 160. Intercalators insert between adjacent base pairs in DNA Ethidium bromide being fluorescent compound is used for visualization of DNA after its electrophoretic separation. Daunorubicin(Intercalator) is used as anticancer agent.
  161. 161. DNA damage induced by base analogs (Bromodeoxyuridine BroU) Base analogs such as Bromodeoxyuridine (BroU) which is analog of T which is incorporated into DNA instead of T. It mispairs with G instead of A, resulting in AT to GC transition.
  162. 162. DNA Repair mechanisms
  163. 163. Repair of thymine- thymine dimer by photo reactivation ❖Photo reactivation repairs UV induced cyclobutane thymine- thymine dimers. This is accomplished by the enzyme DNA photolyase, which contains two chromophores capable of absorbing light. 1. N5N 1 methenyltetrahydrofolyl polyglutamate( MTHF poly Glu) 2. FADH ➢The enzyme DNA photolyase uses energy derived from absorbed light to reverse damage.
  164. 164. DNA Repair by enzyme DNA photolyase using photo reactivation Two chromophores of DNA photolyase viz. N5N 1 methenyltetrahydrofolyl polyglutamate(MTHF poly Glu) and FADH are capable of absorbing light. The enzyme DNA photolyase uses energy derived from absorbed light to reverse damage.
  165. 165. Types of Excision Repair for DNA ❖Types of Excision Repair for DNA are : 1. Base Excision Repair for DNA 2. Nucleotide Excision Repair for DNA
  166. 166. Base Excision Repair of DNA ❖Base Excision Repair of DNA occurs as follows : 1. Deamination of cytosine ,Adenine and guanine to uracil, hypoxanthine and xanthine respectively. 2. The bases are removed by DNA Glycosylases to generate apurinic and apyrimidinic sites. 3. An endonuclease cleaves the a basic sugar and phosphate. 4. The gap is repaired by DNA pol I and sealed by ligase.
  167. 167. Base Excision Repair of DNA ThebasesareremovedbyDNAGlycosylasestogenerateapurinicandapyrimidinicsites.Anendonuclease cleavestheabasicsugarandphosphate.ThegapisrepairedbyDNApolIandsealedbyligase.
  168. 168. Nucleotide Excision Repair for DNA ❖Nucleotide Excision Repair (NER) of DNA : Lesions such as thymine-thymine dimers , base adducts and 6,4 photoproducts are repaired by Nucleotide Excision Repair. • Upto 3 bp of damaged DNA is replaced by Excision of a short stretch of Nucleotides on either site of the lesions. • Two phosphodiester bonds on the strand containing defect are hydrolyzed by an Excision nuclease termed as Excinuclease present in both E.coli and human . The gap is repaired by DNA pol I in E.coli and pol / in human. • The Nick is sealed by DNA ligase.
  169. 169. Nucleotide Excision Repair for DNA NucleotideExcisionRepair(NER) ofDNA:Lesionssuchasthymine-thyminedimers,baseadductsand6,4 photoproductsarerepairedbyNucleotideExcisionRepair.
  170. 170. Nucleotide Excision Repair for DNA in E.Coli and Human Upto3bpofdamagedDNAisreplaced byExcisionofashortstretchof Nucleotidesoneithersiteofthe lesions.TwophosphodiesterbondsonthestrandcontainingdefectarehydrolyzedbyanExcisionnuclease termedasExcinucleasepresentinbothE.coliandhuman.ThegapisrepairedbyDNApolIinE.coliandpol/ inhuman. TheNickissealedbyDNAligase.
  171. 171. Repair mechanism for DNA mismatches :1 ❑The DNA mismatch repair (MMR) system corrects errors made during DNA replication to ensure high degree of fidelity of replication . ❑ DNA mismatches arise due to 1. Mispairing 2. Insertion of extra unpaired bases 3. Deletion as a result of polymerase slippage Before DNA mismatches are repaired, the parental (template ) strand is methylated on the N5 position of all adenine residues within the 5‘GATC3’ sequences by Dam methylase in order to differentiate from newly synthesized strand . The DNA mismatch repair is targeted to the transiently unmethylated newly synthesized strand. This system repairs up to 1 bp from hemi methylated GATC sequence.
  172. 172. Strand directed mismatch repair for DNA in E.Coli • Specific proteins scan the newly synthesized DNA Strand (new DNA Strand is identified by not being methylated). • The mismatch area is identified ,a loop is made. • In E.coli ,this recognition and looping is made by three proteins viz MutS, MutH , MutL .Then that segment are removed. Finally the correct segments are synthesized by DNA polymerase, SSBs and ligase .
  173. 173. Repair mechanism for DNA mismatches in E.Coli In E.coli ,the proteins required to recognize the mutation and catalyze nicking include MutS, MutH , MutL . MutS recognizes mismatch and repairs it. MutH attaches to MutS by MutL and cleaves unmethylated newly synthesized strand by site specific endonuclease activity marking strand for repair . An endonuclease degrades the unmethylated strand containing mutation from the GATC through the mutation to remove damaged DNA. If mutation is present between two GATC sites ,the DNA between two sites removed, the gap is filled by polymerase and nick is sealed by ligase . Several thousands of bases may be replaced by in ode to correct one mismatch.
  174. 174. Strand directed mismatch repair of DNA in E.Coli The mismatch area is identified ,a loop is made. In E.coli ,this recognitionand looping is made by three proteinsviz MutS,MutH, MutL.Thenthatsegmentareremoved.Finallythecorrectsegmentsare synthesizedbyDNApolymerase,SSBsandligase.
  175. 175. Repair mechanism for DNA mismatches in human ❖Repair mechanism for DNA mismatches in human: ➢hMSH2 and hMLH 1 are human analogs of E.coli MutS, and MutL . ➢ In human defective mismatch repair in Hereditary nonpolyposis colon cancer (HNPCC) is associated with microsatellite instability .
  176. 176. Transcription coupled Repair ❑Transcription coupled Repair : The template strand of transcribed region responds immediately by stalling of RNAP at the site of damage .The enzyme backs away. The Repair proteins are recruited and the defect is repaired by NER. ❖ Cockyane ’s syndrome results from deficiency of proteins that help to recognize the halted RNAP.
  177. 177. Role of RPNA and Repair proteins at the DNA damage site in human Transcription coupledRepair:ThetemplatestrandoftranscribedregionrespondsimmediatelybystallingofRNAPatthesiteof damage.Theenzymebacksaway.TheRepairproteinsarerecruitedandthedefectisrepaired byNER.
  178. 178. Failure of Transcription coupled Repair in Cockyane’ssyndrome ThetemplatestrandoftranscribedregionrespondsimmediatelybystallingofRNAPatthesiteofdamage.The enzymebacksaway.TheRepairproteinsarerecruitedandthedefectisrepaired byNER.Cockyane’s syndromeresultsfromdeficiencyofproteinsthathelptorecognizethehaltedRNAP.
  179. 179. ClinicalmanifestationsofCockyane’ssyndrome
  180. 180. Summary of Repair mechanisms for DNA Mechanisms for DNA Repair Defect Repair 1.Mismatch Repair Copying error 1-5 bases unpaired strand cutting and endonuclease digestion 2.Nucleotide Excision Repair for DNA Chemical damage to a segment 30 bases removed and then correct bases added 3.Base Excision Repair Chemical damage to single base bases removed by N – glycosylase and then new bases added 4.Double strand break free Radicals and radiation Unwinding , alignment ,ligation
  181. 181. Types of DNA repair systems in E.Coli DNArepairsystemsinE.Coli TypeofDNAdamage EnzymesandproteinsinvolvedinDNA repair Mismatchrepair Mismatchescopyingerrors DNAmethylase MutH,MutL,MutSproteins DNAhelicaseII SSB DNApolymeraseIII ExonucleaseI DNAligase Baseexcisionrepair Spontaneous,chemicalorradiationdamagetoa singlebase,Pyrimidinedimers,alkylatedbases DNAglycosylases APendonucleases DNApolymerasesI DNAligase Nucleotideexcisionrepair DNAlesionsthatcauselargestructuralchanges e.g. pyrimidine dimersmaybeduetochemical ,radiationorspontaneous. ABCExcinuclease DNApolymeraseI DNAligase Directrepair Pyrimidinedimers,O6 –methylguanine DNAphotolyases,O6 –methylguanine
  182. 182. Summary of DNA damage and repair
  183. 183. Consequences of unsuccessful DNA damage repair 1. Cell death due to Mutations→ Apoptosis , Cell necrosis 2. Cell which survives after mutation→ premature differentiation ,malignant transformation, senescence
  184. 184. Diseases associated with defective DNA Repair mechanisms Diseases Defectss Xerodermapigmentosa(XP) Defective NucleotideExcisionRepair(NER) forDNA,sensitivitytoUVlight,skin cancer AtaxiaTelangiectasia DefectiveATMgene,sensitivitytoUVlight,lymphoreticularneoplasm Fanconi’sanemia Defectivegenesareinchromosome20qand9q.defectinDNAcrosslink Repair Bloom’ssyndrome geneareinchromosome15q,defectinDNAligaseorhelicase, lymphoreticularmalignancies Cockyane’ssyndrome DefectiveNucleotideExcisionRepair(NER) forDNA,transcriptionfactorIIHis defective,stuntedgrowth,mentalretardation Hereditarynonpolyposis coloncancer(HNPCC) = Lych’ssyndrome Defectivegene inchromosome2.DefectinhMSH1and2genes,mismatch RepairmechanismforDNAisdefective.
  185. 185. Xeroderma pigmentosa • DNA is constantly being subjected to environmental insults that cause the alteration of nucleotide bases. • Exposure of cells to UV light can result in covalent joining of adjacent thymine (thymine-thymine dimer) . Luckily ,cells are remarkable efficient at repairing the damage done together DNA. • In a rare genetic disease Xeroderma pigmentosa the thymine dimer formed in DNA after skin cells are exposed to UV light and cannot be repaired. Patients suffer from skin cancer .The enzyme required in repair mechanism ,viz UV specific endonuclease is absent in these individuals .
  186. 186. Xeroderma pigmentosa InararegeneticdiseaseXerodermapigmentosathethyminedimerformedinDNAafterskincellsare exposedtoUVlightandcannotberepaired.Patientssufferfromskincancer.Theenzymerequiredinrepair mechanism,vizUVspecificendonucleaseisabsentintheseindividuals.
  187. 187. Ataxia Telangiectasia ❖Ataxia Telangiectasia : 1. Common autosomal recessive disease 2. sensitivity to UV light 3. Cerebellar ataxia 4. Telangiectasia in eyes 5. lymphoreticular neoplasm 6. Ataxia Telangiectasia mutated (ATM) gene located at chromosome 11 q mutated (ATM gene 1% of total population). 7. Disease is manifested in 1:40000 persons.
  188. 188. Ataxia Telangiectasia mutated (ATM) gene located at chromosome 11 q
  189. 189. Clinical manifestations of Ataxia Telangiectasia Commonautosomalrecessivedisease,sensitivitytoUVlight,Cerebellarataxia,Telangiectasia(spiderveins) ineyes,lymphoreticularneoplasm
  190. 190. Mutations • Mutations are some defects remaining in DNA after proofreading in DNA replication . • Mutations may result from faulty DNA replication or repair. • Mutation rate is 6 nucleotide changes /year in the germ cell lines of an individual . • Out if every 10 6 cell divisions ,one Somatic Mutation taking place .
  191. 191. Mutations ❖Mutation is a change in the nucleotide sequence of a gene ( a portion DNA ). ❖Mutation will result in substitution of different amino acid at the corresponding site in the protein molecule. ❖The altered protein may 1. have a function similar to the normal protein. 2. have a partial but abnormal function or may 3. not be capable of functioning at all. These three possibilities are exemplified by hemoglobinopathies *. ❖Natural Mutations are responsible not only for genetic disorders ,but also for the evolutions of newer and novel forms of life .This is suggested to be responsible evolution.
  192. 192. Mutations *related hemoglobin synthesis Functional Status of hemoglobin Proteinhemoglobin Aminoacidsubstitution Codons function not altered Hb ,  chain Hb Hikari 61 Lysine Asparagine AAA or AAG AAU or AAC function partially altered Hb ,  chain Hb S 6 Glutamic acid Valine GAA or GAG GUA or GUG Non Functional Hb ,  chain Hb M ( Boston) 58 Histidine Tyrosine CAU or CAC UAU or UAC
  193. 193. Inhibitors of DNA Replication as antibacterial agents Some drugs inhibit bacterial enzymes and they will arrest new DNA synthesis and arrest the bacterial cell division. These drugs are useful as antibacterial agents(will not affect human cells). Antibacterial agents (Drugs) functioning as the Inhibitors of DNA Replication enzyme - Bacterial DNA gyrase Ciprofloxacin Nalidixic Acid Novobiocin
  194. 194. Ciprofloxacin as Antibacterial agent (Drug) functioning an inhibitor of DNA Replication enzyme -Bacterial DNA gyrase CiprofloxacininhibitssbacterialDNA gyrase enzyme.ItwillarrestnewDNAsynthesisandarrestthe bacterialcelldivision.Ciprofloxacin, Nalidixic Acid and Novobiocin areusefulasantibacterialagents(will notaffecthumancells).
  195. 195. Inhibitors of DNA Replication as anticancer agents Some drugs inhibit human enzymes and the will arrest new DNA synthesis and arrest the cell division. These drugs are useful as anticancer agents . Anticancer agents(Drugs) functioning as Inhibitors of human DNA Replication enzyme(DNA Topoisomerases)/DNA polymerase/thymidylate synthase Doxorubin inhibit human DNA Topoisomerases Andriamycin inhibit human DNA Topoisomerases Etoposide inhibit human DNA Topoisomerases 5-Fluorouracil inhibit human DNA polymerase Mercaptopurine inhibit thymidylate synthase
  196. 196. Doxorubinfunctioningas InhibitorofhumanDNAReplicationenzyme(DNATopoisomerases)
  197. 197. Mechanism of action of 5-Fluorouracil as an Anticancer agent(Drug) Antimetabolitessuchas5-FluorouracilandmethotrexateinhibittheformationofdNTPs,thesubstrateforreplication.
  198. 198. Inhibitors of DNA Replication ❖Certain nucleotide analogues like arabinose cytosine and arabinose adenine which contain Arabinose ( another pentose) instead of the usual ribose inhibit synthesis of DNA. • Arabinose cytosine : used as an anticancer agent • Arabinose adenine : used as an antiviral agent ❖Antimetabolites such as 5-Fluorouracil and methotrexate inhibit the formation of dNTPs, the substrate for replication. ❖Substrate analogs are incorporated into DNA polymerization .These include a) Azothymidine (AZT ,zidovudine) used in treatment of human immunodeficiency virus (HIV) infection b) Cytosine Arabinoside (cytarabine),an antileukemia drug. ❖ Direct Inhibitors of DNA Replication include
  199. 199. Mechanism of action Cytosine Arabinoside as an antileukemia drug Cytosine Arabinoside is a substrate analogs .It is incorporated into DNA and inhibits DNA replication/ polymerization. Thus it arrests cell division(functions an antileukemia drug).
  200. 200. Direct Inhibitors of DNA Replication ❖ Direct Inhibitors of DNA Replication include: • Intercalators containing aromatic rings that insert between adjacent base pairs in DNA . These include anticancer agents such as daunorubicin, produced by strains of Streptomyces. • DNA damaging drugs including a) Alkylating agents that are strong electrophiles that bind to DNA. Examples include methylmethane sulphonate (MMS) and ethyl nitrosourea. b) Platinum coordination complexes such as cisplatin that interact with the N7 Guanine in DNA to form crosslinks between adjacent Guanines. c) Bleomycin produced by Streptomyces verticillus cause DNA damage by interacting with O2 and Fe 2+ .These are used as cancer chemotherapeutic drugs .
  201. 201. Synthetic drugs as Inhibitors of DNA Replication in Prokaryotes • Mitomycin C , Novobiocin : Inhibitors of DNA replication enzyme DNA polymerase and hence cell division→ Inhibit cell division) • Ciprofloxacin, ,Nalidixic Acid (Mechanism of action : inhibition of DNA Topoisomerase II(DNA Gyrase )→DNA replication inhibited → bacterial cells multiplication stops. • 5 Fluorouracil & Mercaptopurine: nucleotide analogs→ used cancer treatment • Doxorubin ,Andriamycin ,Etoposide : inhibit human DNA Topoisomerases → used cancer Treatment
  202. 202. Inhibitors of DNA Replication enzymes Inhibitors of DNA Replication enzymes such as nalidixic acid and fluoroquinolone are used in the treatment of urinary tract infections . These inhibit bacterial topoisomerase II( DNA gyrase). It prevents from relieving positive supercoils during DNA Replication.
  203. 203. Plasmids • Plasmids are small circular DNA molecules present in some bacteria besides their large circular chromosomal DNA. • chromosomal DNA of bacteria has complete set of genes for the existence and propagation of the bacteria.  • Plasmids have sufficient genetic information to replicate in the bacterial cells and synthesize few proteins . • Some naturally occurring bacterial plasmids code for enzymes proteins that can protect the cells from bactericidal effects of antibiotics. Thus bacteria which have such plasmids have an advantage over other bacteria to survive even in the presence of antibiotics .
  204. 204. Plasmids  Plasmids are relatively smaller DNA molecules ,they can be easily isolated from bacterial cell debris and chromosomal DNA with considerable ease .Therefore Plasmids are used as carriers / vectors of DNA segment from an other source to generate recombinant DNA . Some naturally occurring bacterial plasmids code for enzymes proteins that can protect the cells from bactericidal effects of antibiotics. Thus bacteria which have such plasmids have an advantage over other bacteria to survive even in the presence of antibiotics .
  205. 205. Use of Plasmids in molecular biology • Since Plasmids are relatively smaller DNA molecules ,they can be easily isolated from bacterial cell debris and chromosomal DNA with considerable ease .Therefore Plasmids are used as carriers / vectors of DNA segment from an other source to generate recombinant DNA . • Such recombinant DNA could be replicated /multiplied / generated in bacterial cells in large quantities for DNA sequencing ,mutagenesis and other molecular biology experiments .
  206. 206. Plasmid Vectors as the tool for genetic engineering Recombinant DNA could be replicated /multiplied / generated in bacterial cells in large quantities for DNA sequencing ,mutagenesis and other molecular biology experiments .

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