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Replication
This document summarizes the process of DNA replication. It begins with parent DNA unwinding and separating into two strands. Each strand then serves as a template for new strand synthesis in a semi-conservative manner. In prokaryotes, replication occurs bidirectionally from a single origin of replication. Eukaryotes have multiple origins of replication to allow for simultaneous replication bubbles. The document discusses enzymes involved like DNA polymerases and primase, as well as mechanisms like Okazaki fragment formation and resolution. It concludes by describing the unique process of telomere replication through the reverse transcriptase telomerase to overcome the end-replication problem.
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Replication
- 1. Replication R. C. Gupta Professorand Head Department of Biochemistry National Institute of Medical Sciences Jaipur, India
- 2. Nucleic acids areknown as information molecules They are of two types − deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) DNA stores genetic information
- 3. The total geneticmaterial present in an organism is known as its genome When a cell divides, the genomic infor- mation is passed on to the daughter cells The DNA transmitted to daughter cells is an exact replica of the DNA of parent cell
- 4. Parent DNA New DNANew DNA Identical base sequence
- 5. Since new DNAis a replica of parent DNA, DNA synthesis is called replication For replication, the two strands of DNA separate Each strand serves as a template for the synthesis of a new strand Replication – An overview
- 6. The new strandis complementary in base sequence to its template strand The template strand and new strand wind around each other Thus, the new DNA is made up of one parent strand and one new strand Therefore, replication is said to be semi- conservative
- 7.
- 8. Replication has beenstudied extensively in prokaryotes Much of the information has been obtained from E. coli Eukaryotic replication is more complex but there are many common features Replication
- 9. For replication, DNAhas to uncoil and the strands have to separate Separation of strands is known as melting or denaturation of DNA This requires breaking of hydrogen bonds between base pairs i.e. A & T and G & C Prokaryotic replication
- 10. There are twohydrogen bonds between A & T and three between G & C Melting of DNA is easier in those regions where A & T pairs are more in number A − S − P − S − P −S − P − − S − P − S − P −S − P − A T T G C
- 11. Replication of DNAbegins at a specific site, called origin of replication (ori) Origin of replication is a site rich in A&T pairs This makes strand separation easier
- 12. In E. coli,the origin of replication (oriC) is a 245-bp sequence There are three tandem repeats of a 13- bp sequence There are four copies of a 9-bp sequence
- 13. oriC (245-bp) Tandem 13-bp sequences (GATCTNTTNTTTT) Four9-bp sequences (TTATNCANA) Origin of replication in E.coli
- 14. In prokaryotes, thereis only one origin of replication Separation of strands at this site produces a fork-like structure This is known as replication fork Replication fork
- 15.
- 16. Both the strandsof DNA are replicated simultaneously The strands are anti-parallel i.e. they run in opposite directions However, nucleic acids are synthesized only in 5’ 3’ direction
- 17. One DNA strandis known as the leading strand The other strand is known as the lagging strand Leading strand is replicated continuously Lagging strand is replicated discontinuously
- 18. The replicating enzymefirst replicates the leading strand Then it turns back and replicates the lagging strand Thus, the overall direction remains 5’ 3’
- 19. Movement of fork New strand(continuous) New strand (discontinuous) Parent strands 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’
- 20. DNA is apolymer of deoxyribonucleotides Deoxyribonucleotides are polymerized by DNA polymerase Primers and Okazaki fragments EMB-RCG However, DNA polymerase has a major limitation
- 21. EMB-RCG DNA polymerase cannotadd a nucleotide to another nucleotide It can add a nucleotide only to an oligonucleotide
- 22. DNA polymerase cannot adda nucleotide to another nucleotide DNA polymerase can add a nucleotide only to an oligo- nucleotide Mononucleotide Mononucleotide Mononucleotide Oligonucleotide
- 23. Thus, the firstrequirement for DNA synthesis is to form an oligonucleotide DNA polymerase cannot synthesize the oligonucleotide To solve this problem, the synthesis of DNA begins with ribonucleotides
- 24. RNA polymerase canadd a nucleotide to another nucleotide Ribonucleotides are polymerized by RNA polymerase RNA polymerase differs from DNA poly- merase in one important respect
- 25. Hence, some ribonucleotidesare poly- merized first to form an RNA primer Synthesis of RNA primer is catalysed by primase Primase is an RNA polymerase
- 26. RNA primer isan oligonucleotide DNA polymerase can add deoxyribonucleo- tides to this oligonucleotide Thus, RNA primer is required to initiate replication
- 27. The substrates forprimer synthesis are ATP, GTP, CTP and UTP The a-phosphate of new ribonucleotide binds to 3’-OH group of the previous one The b- and g-phosphates are split off in the form of inorganic pyrophosphate
- 28. RNAP OH H OH H HH O CH2 O P O H OH H HH O P ~ P O P OH H OH H HH OO P Basen+1 3’ OHOH H H H O P ~ P ~ P – O– CH2 Formation of phosphodiester bond between the last nucleotide and the new nucleotide abg CH2 CH2 Basen+1 Basen Basen 5’ 5’ 5’ 5’ 3’ 3’ 3’ New nucleotide Last nucleotide H
- 29. Selection of nucleotidesis governed by the base sequence of template strand The nucleotides are selected according to the base-pairing rule i.e. Adenine opposite thymine Uracil opposite adenine Guanine opposite cytosine Cytosine opposite guanine
- 30. After formation ofRNA primer, deoxyribo- nucleotides are added by DNA polymerase The substrates are dATP, dGTP, dCTP and dTTP The reaction is similar to the addition of ribonucleotides This process continues until about 100 deoxyribonucleotides have been added
- 31. The resulting polynucleotideis known as an Okazaki fragment It is made up of a short RNA primer and several deoxyribonucleotides Okazaki fragment 3’5’ Primer Deoxyribonucleotides
- 32. Several Okazaki fragmentsare formed on the template strand 3’ DNA template 3’ Okazaki fragments 5’ 5’ After replication of several leading and lagging strands, RNA primers are removed
- 33. Ribonucleotides are removedone by one from the 5’-end They are replaced by deoxyribonucleotides according to the base-pairing rule The remaining portions of parent DNA are then replicated The different DNA fragments are sealed together
- 34. Each new strandforms a duplex with the template strand of the parent DNA The winding begins even while the replication is going on
- 35. Enzymes and proteinsinvolved in prokaryotic replication The process of replication requires a number of enzymes and protein factors In E. coli, the unwinding of parent DNA is initiated by dna B protein
- 36. First, dna Aprotein binds to the DNA at the initiation site Then, it is joined by dna B and dna C proteins The dna B protein begins the unwinding
- 37. As replication proceeds,further unwinding is catalysed by helicase (rep protein) ATP is required as a source of energy for unwinding DNA The unwound strands are held apart by single strand binding (SSB) protein
- 38. Synthesis of RNAprimer is catalysed by primase Deoxyribonucleotides are added to the RNA primer by DNA polymerase There are three DNA polymerases in E. coli
- 39. Arthur Kornberg haddiscovered the first DNA polymerase (DNA polymerase I) Arthur Kornberg DNA polymerase II and DNA polymerase III were discovered later
- 40. Deoxyribonucleotides are addedto RNA primer by DNA polymerase III holoenzyme DNA polymerase III possesses two different catalytic activities: Polymerase activity 3’ 5’ Exonuclease activity
- 41. The polymerase activityadds deoxyribo- nucleotides The 3’ 5’ exonuclease activity is meant for proof-reading and error-correction
- 42. The enzyme movesalong the template strand The polymerase activity add a deoxyribo- nucleotide After this addition, the enzyme moves back by one nucleotide distance It checks the new base for correctness of base-pairing
- 43. If the base-pairingis correct, the enzyme moves ahead It adds another deoxyribonucleotide The replication continues
- 44. If a wrongbase is detected, the last nucleotide is split off The splitting is catalysed by 3’ 5’ exo- nuclease activity of the enzyme The correct nucleotide is, then, added by the polymerase activity This ensures a high fidelity in replication
- 45.
- 46. The ribonucleotides ofRNA primer are removed and replaced with deoxyribo- nucleotides by DNA polymerase I DNA polymerase I possesses three different catalytic activities: 5’3’ Exonuclease activity Polymerase activity 3’ 5’ Exonuclease activity
- 47. 5’ 3’Exonuclease activity removes the last ribonucleotide from 5’-end of primer In its place, a deoxyribonucleotide is added by the polymerase activity 3’ 5’ Exonuclease activity checks correctness of base-pairing and removes wrong nucleotides
- 48. The DNA fragmentsare joined together by DNA ligase Template strand and the new strand are wound around each other by DNA gyrase DNA polymerase II is a DNA repair enzyme
- 49. The overall processof replication is similar to that in prokaryotes Many enzyme and protein factors involved in replication are identical But there are some distinct differences Eukaryotic replication
- 50. The differences have arisendue to: Bigger size of DNA More intricate cellular architecture More complex cell biology in eukaryotes
- 51. Eukaryotic DNA ismuch bigger than pro- karyotic DNA For example, human genome is 800 times as big as the E. coli genome If there is only one site of origin, repli- cation of human DNA will take a long time Replication bubbles
- 52. For speedy replication: EukaryoticDNA has several sites of origin The template DNA is nicked at a number of places DNA is unwound at several places Replication begins simultaneously at these sites
- 53. EMB-RCG The unwound portionsform a number of replication bubbles which are replicated simultaneously Origin Origin Replication bubbles
- 54. Eukaryotes have moreDNA polymerases than prokaryotes Some enzymes are unique to eukaryotes Eukaryotic enzymes
- 55. The enzymes uniqueto eukaryotes are: • DNA polymerase a • DNA polymerase b • DNA polymerase g • DNA polymerase d • DNA polymerase e • DNA topoisomerase II • Telomerase
- 56. DNA polymerase a DNApolymerase a synthesizes the lagging strand, and possesses: Primase activity DNA polymerase activity 3’ 5’ Exonuclease activity in some species
- 57. DNA polymerase bis similar to DNA polymerase II of prokaryotes It is a DNA repair enzyme DNA polymerase b
- 58. In eukaryotes, someDNA is present in mitochondria also DNA polymerase g is a mitochondrial enzyme It replicates mitochondrial DNA DNA polymerase g
- 59. DNA polymerase d DNApolymerase d synthesizes the leading strand It possesses: DNA polymerase activity 3’ 5’ Exonuclease activity
- 60. DNA polymerase e DNApolymerase e is similar to DNA poly- merase I of prokaryotes and possesses: 5’ 3’ Exonuclease activity Polymerase activity 3’ 5’ Exonuclease activity
- 61. DNA topoisomerase IIis similar to DNA gyrase of prokaryotes It winds the template strand and the new strand around each other DNA topoisomerase II
- 62. The ends ofchromosomes are known as telomeres Telomerase is required for replication of telomeres Telomerase
- 63. Telomere is arepeating sequence of bases (GGGTTA) Telomere is present at the ends of eukaryotic chromosomes The human telomere can have a length of 100-15,000 base pairs Telomere
- 64. DNA polymerase canadd nucleotides only to an oligonucleotide When replication machinery reaches the telomere, bases left in the template strand are too few to form the RNA primer The end replication problem in eukaryotes
- 65. DNA polymerase cannot jointhese No place for a primer ∕ ∕
- 66. Therefore, the terminalpart of lagging strand cannot be replicated Since the terminal part is not replicated, the telomere becomes shorter This happens at the time of every cell division
- 67. Each time acell divides, some telomere is lost When the length of telomere decreases below a critical limit, DNA cannot be replicated The cell undergoes apoptosis
- 68. Telomere shortening canbe prevented by adding some nucleotides to the telomere The nucleotides are added at the 3’-end of the template strand As a result, the 3’-end becomes long enough for primer synthesis The solution
- 69. Addition of nucleotidesrequires a special polymerase and a template Polymerase activity and the template are present in a single conjugated protein The conjugated protein is telomerase
- 70. Telomerase is alsoknown as telomere terminal transferase It is made up of a protein and a RNA The protein part possesses polymerase activity The RNA component acts as a template Telomerase
- 71. A few basesof the RNA component hybridise with complementary bases of telomere The remaining part of RNA acts as a template The polymerase activity adds some nucleotides to the 3’-end
- 72. Telomerase adds complementarydeoxyribo- nucleotides to the 3’-end of the telomere ----TTA3’ End of telomere RNA template AAUCCCAAU ----TTAGGGTTA AAUCCCAAU 3’ End of telomere RNA template Telomerase
- 73. Telomerase dissociates andtranslocates towards the new 3’-end RNA component binds to the new 3’-end, and the telomere is extended again This cycle is repeated several times
- 74. −−−TTAGGGTTA Telomerase binding AAUCCCAAU −−−TTAGGGTTAGGGTTA AAUCCCAAU −−−TTAGGGTTAGGGTTA Telomere extension 3’End of telomere Telomerase translocation AAUCCCAAU −−−TTAGGGTTATelomere end Telomere end Telomere end
- 75. When the telomerebecomes sufficiently long, the telomerase leaves it A primer is synthesized opposite the 3’- end of telomere Deoxyribonucleotides are added to the primer in the usual way The end of the telomere is replicated
- 76. During foetal life,telomerase is present in all the cells After birth, it is present only in germ cells Telomerase activity is lost in somatic cells during development
- 77. But telomerase activityreturns in cancer cells Therefore, there is no shortening of telomeres in cancer cells Cancer cells can go on dividing for ever
- 78. Replication of DNAis vital for life; inhibition of replication prevents cell division Inhibitors of eukaryotic replication can be used as anti-cancer drugs They will check the multiplication of cancer cells Inhibitors of replication
- 79. Cisplatin produces intra-strandpurine cross-links and prevents replication Mitomycin C produces intra-strand G−G and inter-strand C−G cross-links, and blocks replication Daunorubicin intercalates in DNA and prevents access to DNA polymerase
- 80.
- 81. Inhibitors acting selectivelyin prokaryotes can be used as antibiotics Antibiotics exploit the differences in pro- karyotic and eukaryotic machinery If a compound inhibits a prokaryotic enzyme but not its eukaryotic counterpart, it can be used as an antibiotic
- 82. Floxacin family ofdrugs (e.g. norfloxacin, ciprofloxacin etc) inhibits DNA gyrase DNA gyrase is present in prokaryotes only The corresponding human enzyme is DNA topo-isomerase II
- 83. The floxacins inhibitDNA gyrase but not DNA topo-isomerase II Being selective inhibitors of prokaryotic replication, floxacins are used as antibiotics Some other inhibitors of DNA gyrase are novobiocin and nalidixic acid
- 84. Baltimore Temin Dulbecco Reversetranscription They named it as RNA-dependent DNA polymerase Baltimore, Temin and Dulbecco dis- covered an unusual DNA polymerase
- 85. RNA-dependent DNA polymeraseis present in some RNA viruses This enzyme can synthesize DNA using RNA as a template When such a virus infects a cell, the viral enzyme synthesizes a DNA strand using its own RNA as a template
- 86. Using the newDNA strand as a template, a second strand of DNA is synthesized The two strands wind around each other forming a double-stranded DNA This double-stranded DNA is a copy of the viral RNA genome
- 87. The DNA copyof viral genome is incor- porated in the genome of the infected cell Incorporation is catalysed by integrase, a viral enzyme Thus, viral genome becomes an integral part of host cell genome
- 88.
- 89. The infected cellbegins to transcribe and translate viral genes Viral proteins are synthesized by the infected cell The proteins surround the RNA transcripts of the viral genome This results in the formation of new viruses
- 90. RNA-dependent DNA polymerasecatalyses reverse of the normal transcription Hence, it is also known as reverse trans- criptase Viruses possessing reverse transcriptase are known as retroviruses
- 91. Human immunodeficiency virus(HIV) is an example of a retrovirus HIV infects helper T cells of human beings and cripples the immune system Reverse transcriptase is used as a tool in recombinant DNA technology