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DNA replication (Introduction, methods, biochemistry, steps involved, prokaryotic DNA replication, Eukaryotic Replication).pptx

This PPT inlcudes, 1. Introduction to DNA replication 2. Methods of replication 3. Semiconservative replication 4. Meselson and Stahl experiment 5. Taylor's experiment 6. Replicon and origin of replication 7. DNA replication in prokaryotes 8. Biochemistry of DNA replication 9. Common enzymes involved in DNA replication 10. DNA polymerase 11. Components of DNA Polymerase III 12. Steps involved in prokaryotic DNA replication 13. DNA replication in eukaryotes 14. Unique aspects of eukaryotic DNA replication 15. Eukaryotic DNA Polymerases 16. Proof reading 17. Comparison between DNA replication in Prokaryotes and Eukaryotes

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Rashmi M G DNA replication 1. Introduction to DNA replication 2. Methods of replication 3. Semi conservative replication 4. Meselson and Stahl experiment 5. Taylor's experiment 6. Replicon and origin of replication 7. DNA replication in prokaryotes 8. Biochemistry of DNA replication 9. Common enzymes involved in DNA replication 10. DNA polymerase 11. Components of DNA Polymerase III 12. Steps involved in prokaryotic DNA replication 13. DNA replication in eukaryotes 14. Unique aspects of eukaryotic DNA replication 15. Eukaryotic DNA Polymerases 16. Proof reading 17. Comparison between DNA replication in Prokaryotes and Eukaryotes
Rashmi M G DNA replication: • Transmission of chromosomal DNA from generations to generations • Achieved when chromosomal DNA is accurately replicated • Pairing AΞT, G=C-important role in replication • Providing two copies of the entire genome • For distribution into each daughter cells 2 strands of parental double helix of DNA separated Base sequence of each parental strand –serve as template Synthesis of new complementary strand 2 identical progeny double helices Parental double helix is half conserved (each parental strand (single) remaining intact) Semi conservative replication Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 6
Rashmi M G Methods of replication Conservative replication Whole original double helix acts as template for a new one One daughter molecule would consist of original parental DNA 2nd daughter had totally new DNA Dispersive replication Some parts of double helix are conserved and some parts are not Parental double strand helix, broken into double stranded DNA segments Synthesis of new double stranded DNA segment= same as conservative Semi conservative replication Parental double helix is half conserved, each parental single strand remaining intact Each parental strand acts as template
Rashmi M G Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 222
Rashmi M G Meselson and Stahl experiment Aim : To Demonstrate of semi conservative replication of DNA in E coli E coli cells Grown in Medium of Nitrogen source (15 N labeled NH4Cl- heavy isotopes of nitrogen) E coli cells culture Transferred to medium of nitrogen source (14 N labeled NH4Cl- light isotopes of nitrogen Allowed to grow continuously Samples were harvested at regular intervals DNA extraction and Isolation its buoyant density determined by centrifugation in CsCl density gradient Showed single band in density gradient
Rashmi M G Source: Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.272,273
Rashmi M G Result: Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 224 Conclusion: DNA replication follows Semi conservative method
Rashmi M G Taylor’s experiment Aim: to suggest and conclude the semi conservative method of replication with the help of Vicia faba Root tips of Vicia faba ( broad beans) Chromosomes isolated and duplicated with thymidine labeled Observed in autoradiography Allowed to duplicate without labeled thymidine Again observed in autoradiography Conclusion: Proves the semi conservative replication of chromosome in broad bean Result: Source: Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.273
Rashmi M G Replicon and origin of replication • DNA replication starts at particular site- origin of replication • Unit of DNA in which replication starts from an origin and proceeds bidirectionally/ unidirectionally to terminus site- replicon (may be linear / circular) Replicon Monorepliconic Bacterial genome Whole bacterial genome= a single replicon Contains single replication origin Multirepliconic Eukaryotic genome Eukaryotic cells has multiple replication origins on single chromosomes Origin of replication • Cis acting sequence • Fully methylated origin • Can initiate replication • In E coli, the origin of replication is oriC • It has 245 bp of DNA • Contains 2 short repeat motifs (9 nucleotides and 13 nucleotides • 9 Nucleotides repeat, 5 copies of which are dispersed throughout oriC- Binding site for a protein- Dna A • 13 Nucleotides repeat, located at one end of the oriC Sequence In prokaryotes, replicon- circular Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 227
Rashmi M G Common features of replication origins: • Replication origins are unique DNA segments that contain multiple short repeated sequences • Three short repeat units are recognized by multimeric origin binding proteins. • These proteins play a key role in assembling DNA polymerase and other replication enzymes at the sites, where replication begins • Origin regions usually contain AT-rich stretch. This property facilitates unwinding of duplex DNA because less energy is required to melt A-T base pairs Replication fork In each replicon, replication is continuous from origin to terminus Accompanied by movement of replicating point- replication fork Replication may be, unidirectional or bidirectional Unidirectional replication Single replication fork in one direction Special types Chloroplasts, mitochondria and Plasmids Bidirectional replication 2 replication forks move in opposite directions Eukaryotes (common) Bacteria
Rashmi M G Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 229
Rashmi M G DNA replication in Prokaryotes (E coli)
Rashmi M G DNA replication in E coli: • Bidirectional replication • From a single origin –OriC • Replicon- circular with no free ends • Replication of DNA in E coli= Theta replication • 3 steps: • Initiation – recognition of the position on a DNA molecule where replication will begin • Elongation- events occurring at the replication fork, where parent polynucleotides are copied • Termination – occurs when parent molecule has been completely replicated
Rashmi M G Biochemistry of DNA replication: In E coli >20 proteins take part in replication Proteins (Enzymes) Initiation • DnaA • Single stranded DNA Binding Protein (SSB) • DnaC • DnaG (Primase) Elongation • DNA polymerase • SSB • DNA gyrase • DNA ligase Termination • Terminus binding protein (Tus) • DNA Topoisomerase IV
Rashmi M G DNA helicase and primase • Opens up the duplex at the replication fork to provide a single stranded template • Primary replicative helicase- DnaB – binds to and moves on the lagging strand template in the 5’→3’ direction unwinding the duplex as it goes • Requires ATP hydrolysis SSB Protein • Inhibition of reannealing of separated strands • Bindings to both separated strands Primase • Synthesizes a short RNA primer (<15 nucleotides) to prime DNA chain elongation • Primosome- complex between primase and helicase, sometimes with accessory proteins Topoisomerase • Required to relieve the positive super coiling that arises from DNA unwinding • In E coli this role is typically fulfilled by DNA gyrase • In eukaryotes TopoIB DNA Polymerase • Catalyze the synthesis of DNA • Both prokaryotes and Eukaryotes contain multiple DNA dependent DNA polymerase • Termed as DNA Replicase • Performs DNA repair also Common enzymes and proteins involved:
Rashmi M G DNA polymerase Template dependent DNA polymerase Template independent DNA polymerase DNA dependent DNA polymerase RNA dependent DNA polymerase (Reverse transcriptase)
Rashmi M G In Eubacteria, - E coli 5 types of DNA polymerases DNA polymerase I • Kornberg enzyme • Arthur Kornberg enzyme • Monomeric protein • Possess 3 enzymatic activities: • 5’3’ Polymerase activity • 5’3’ exonuclease activity • 3’5’ exonuclease activity • Primer removal function (with 5’3’ exonuclease activity) • Gap filling (5’3’ polymerase activity) and DNA repair DNA polymerase II • Monomeric protein • 5’3’ Polymerase activity • 3’5’ exonuclease activity • Alternative DNA repair polymerase • Can replicate DNA if the template is damaged • Low polymerizatio n rate DNA polymerase III • Primary enzyme • Multiprotein complex • (10 distinct polypeptides) • High polymerization rate • High Processivity DNA polymerase IV • Y family DNA polymerase • dinB • Do not contain 3’5’ exonuclease activity • Low catalytic efficiency • Low Processivity • Low fidelity • Involved in tranlesion synthesis • Replicate damaged DNA by bypassing damaged nucleotides DNA polymerase V • Y family DNA polymerase • Found in bacteria like E coli • Error prone polymerase • Involved in SOS response to extensive DNA damage
Rashmi M G Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 235
Rashmi M G Components of DNA Polymerase III Catalytic core Dimerization component Processivity component Clamp loader α β θ τ Causes 2 catalytic core at replication fork to link together and form asymmetrical dimer Homodimer of β subunits (acts as sliding clamp) 5 subunits subassembly γ δ χ ψ δ϶ 5’3’ Polymerase activity 3’5’ exonuclease activity (Proof reading) To enhance proofreading activity of ϵ ϒ complex Loading of β clamp onto the DNA Facilitated by ϒ complex Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 241
Rashmi M G ENZYME EXONUCLEASE ACTIVITIES Subunits 3’5’ 5’3’ Function DNA polymerase I 1 YES YES DNA repair Gap filling Primer removal DNA polymerase II 1 YES NO DNA repair DNA polymerase III 10 YES NO Main replicating enzyme Major DNA polymerase involved in replication of bacterial genome
Rashmi M G Steps involved in prokaryotic DNA replication
Rashmi M G DnaA protein + oriC (9 mer sequence) =Initial complex (facilitates initial strand separation or melting) Occurs at oriC 13 mers Melting of 2 strands generates unpaired template strands Mediated by DnaB protein- Helicase Requires ATP and forms open complex 1 molecule= hexamer of identical subunit Clamp around each 2 single strand in open complex formed between DnaA and oriC Requires ATP and DnaC acts as helicase loader Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 234 Initiation step: DnaA protein- initiates replication in E coli at oriC
Rashmi M G Helicase Move along DNA duplex Utilize energy of ATP hydrolysis to separate strands SSB Protein Inhibits reannealing by binding to both separated strands Primase Synthesize short primer RNA complementary to both strands of DNA duplex Image showing the importance of SSB protein
Rashmi M G Elongation reaction: Nucleophilic attack by 3’ hydroxyl group of primer on innermost phosphorus atom of deoxyribonucleoside triphosphate Phosphodiester bridge forms= release of pyrophosphate Catalytic metal ions (Mg2+ ) present in active site has important role Metal ions+ 3’OH = Reduce association between O and H leaves a nucleophilic 3’O- After catalysis pyrophosphate product stabilized through similar interaction with metal ions Hydrolysis by pyrophosphatase Metal ions + Triphosphate of incoming dNTP Neutralize their negative charge Helps to drive polymerization forward Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 232 Elongation step: DNA polymerase catalyze step by step addition of deoxyribonucleotide units to DNA chain
Rashmi M G Source: Peter J Russell, iGenetics A molecular approach, 3rd edition, Page no.41 Source: Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.275
Rashmi M G At each growing fork, 1.Leading strand: Synthesized continuously from single primer on leading strand template Growing fork Grows 5’3’ 2.Lagging strand: Complicated, DNA polymerase can add nucleotide only to 3’ end of primer / growing DNA strand Growing fork Leading strand growth Growing fork Leading strand growth Short pieces of DNA= Okazaki fragments – repeatedly synthesized on lagging strand template
Rashmi M G Primase+ helicase = Primosome Makes RNA primer DNA Polymerase III (synthesizing leading strand copy) extends RNA primer Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 237
Rashmi M G Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 331
Rashmi M G As each Okazaki fragment formation completes, the RNA primer of previous fragment is removed by the 5’3’ exonuclease activity of DNA polymerase I This enzyme also fills in the gaps between the lagging strand fragments, which then are ligated together by DNA ligase Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 236
Rashmi M G Termination step: • In bacterial genome, replication is bidirectional from single point • 2 replication fork should meet at a position diametrically opposite the origin of replication • Replication terminates at terminus region Terminus region: • Contains multiple copies of about 23 bp sequences called Ter (for terminus) • Each acting as recognition site for a sequence specific DNA binding protein called Tus (terminus utilization substance) Tus protein+ Ter sequence • Allows replication fork to pass if fork is moving ion one direction • Blocks progress if fork is moving in opposite direction around genome • Directionality is set by orientation of Tus protein on double helix • When approached from one direction- Tus blocks the passage of DnaB Helicase • When approached from other direction- DnaB –able to cross Tus protein Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 332 Completion of replication in circular chromosome, 2 new circles may be physically interlocked/ catenated Separated so that each daughter cell receives single dsDNA upon cell division Decatenation of interlocked circle- By Topoisomerase IV
Rashmi M G Source: Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.274
Rashmi M G DNA replication in Eukaryotes
Rashmi M G To summarize, Steps involved in DNA replication in Prokaryotes 1. DnaA protein+ oriC (9 mer)- Initial complex (ATP NEEDED) 2. DnaB protein- Helicase- for further melting 3. DnaC- Helicase loader- joins initial complex 4. SSB protein – Keeps unwound DNA strand in extended form 5. RNA primase- DnaG- For primer synthesis 6. DNA polymerase- Initiation of DNA polymerization 7. DNA polymerase I- Removal of primer 8. DNA polymerase II- DNA repair activity 9. DNA polymerase III- Main replicating enzyme- chain elongation, proof reading 10.DNA ligase- Joining of fragments (ATP NEEDED) The eukaryotic DNA replication is similar to prokaryotic DNA replication but differences lies in these following aspects, 1. Multiple replication origins in their chromosomes 2. More types of DNA polymerases with different functions 3. Linear DNA replication 4. Nucleosome assembly immediately after DNA replication 5. Different termination strategy
Rashmi M G Unique aspects of eukaryotic DNA replication 1. Multiple replication origins • Eukaryotic genome= greater amount of DNA • Ex. Yeast, Drosophila • Replication takes more time • To facilitate the rapid synthesis of large quantities of DNA, Eukaryotic chromosomes contain multiple replication origins • Origins in yeast- Autonomously replication sequences • Eukaryotic replication origins- not only acts as sites of replication initiation, but also control the timing of DNA replication • These regulatory functions are carried out by a complex of more than 20 proteins, called Pre-replication complex (Pre-RC), which assembles at replication origins. Source: Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.282
Rashmi M G 2. More types of DNA polymerases with different functions Eukaryotic cell contains, Many types of DNA polymerase Present both in nucleus and organelles (Mitochondria and chloroplast) 3 major types of DNA polymerase (present in nucleus for nuclear DNA replication): 3. DNA polymerase α 4. DNA Polymerase δ 5. DNA polymerase ϵ Enzyme Exonuclease activity 3’5’ 5’3’ Function DNA polymerase α NO NO Priming during replication DNA polymerase β NO NO Base excision repair DNA polymerase γ YES NO Mitochondrial DNA replication DNA polymerase δ YES NO Lagging and leading strand synthesis DNA polymerase ϵ YES NO - Major DNA polymerase involved in Replication of Eukaryotic genomes
Rashmi M G Eukaryotic DNA polymerases 1. DNA polymerase α • Unusual polymerase • Has both DNA polymerase and primase activity • 5’3’ DNA dependent DNA polymerase activity • 5’3’ DNA dependent RNA polymerase activity • Heterotetramer Primase activity: • Incorporates NMP- To synthesize short RNA primer (iRNA initiator RNA) • Complement – ssDNA template Polymerase activity: • Adds 20-30 NMP to 3’ end of iRNA (also DNA – iDNA) • No proofreading activity • (absence of 3’ 5’ exonuclease activity Function: • Initiates DNA synthesis on both leading and lagging strand • Polymerase α + initiation complex- at origin: • Synthesize short iRNA followed by 20-30 bases of iDNA • Then replaced by other DNA polymerase- extend the chain • This process is called- polymerase switching
Rashmi M G 2. DNA Polymerase δ • Multi subunit nuclear enzyme • 4 subunits • Has 5’3’ polymerase activity • 3’5’ exonuclease activity • Requires associated 30kDa-protein (proliferating cell nuclear antigen PCNA) function as sliding clamp • For high Processivity • Requires clamp loader- replication factor C acts as clamp loader Function: Alone replicates leading and lagging strands of DNA 3. DNA polymerase ϵ • Multi subunit nuclear enzyme • 4 subunits • 5’3’ polymerase activity • 3’ 5’ exonuclease activity • High processive DNA synthesis with help of sliding clamp and clamp loader • Function: Not known precisely 4. DNA polymerase γ • Sole polymerase • Participate in mitochondrial DNA replication • 3 subunits • 5’3’ polymerase activity • 3’5’ exonuclease activity
Rashmi M G Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 329
Rashmi M G 3. Nucleosome assembly immediately after DNA replication • Eukaryotic DNA is complexed to histone proteins in nucleosome structures that contribute stability and packing of the DNA molecule. • The disassembly and reassembly of nucleosomes on newly synthesized DNA probably takes place in replication, but the precise mechanism for these processes has not yet been determined • Before replication, a single DNA molecule is associated with histone proteins • After replication and nucleosome assembly, two DNA molecules are associated with histone proteins Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 247
Rashmi M G 4. Termination step in Eukaryotic replication 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ RNase H removes the primer, up to the last ribonucleotide 5’ 3’ 3’ 5’ FEN1 removes that last ribonucleotide 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ DNA ligase links the two DNA fragments Helicase breaks the H- Bond between primer and template 5’ 3’ 3’ 5’ Removal of the primer 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ DNA ligase links the two DNA fragments Rnase H model The Flap model FEN1 cuts at the junction
Rashmi M G Proofreading • Needed by mammals ( in large genomes) • Scanning the termini of nascent DNA chain for errors • Correcting them before continuing chain extension • Carried out by 5’3’ exonuclease activity that is built into DNA polymerases • When template- primer DNA has a terminal mismatch (an unpaired/ incorrectly paired base/ sequence of bases at 3’ of primer) • 3’5’ exonuclease activity of DNA polymerase I or II hips off the unpaired base/ bases • When appropriate base- paired terminus is produced • The 5’3’ polymerase activity of enzyme begins resynthesis by adding nucleotides to 3’ end of primer strand Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 339
Rashmi M G Comparisons
Rashmi M G Enzyme/ Protein E coli Human Helicase DnaB MCM ssDNA binding protein SSB RPA (Replication protein A) Primase DnaG Dna polymeraseα/ Primase Replicase DNA polymerase III DNA polymerase ϵ / DNA polymerase δ Topoisomerase Gyrase Topo I, II Processivity component β clamp PCNA (Proliferating cell nuclear antigen) Clamp loader γ-complex RFC (Replication factor C) DNA replicating enzymes/ proteins from E coli and Human
Rashmi M G Quantitative parameters E coli Human DNA content, number of nucleotide pairs per cell 3.9✗ 106 About 109 Rate of replication fork progression per replication (per second) ~1000 ~100 Number of replication origins per cell 1 103 -104 Time required for complete genome replication ~42 minutes ~8hours Comparison of quantitative parameters of DNA replication in E coli and Human

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DNA replication (Introduction, methods, biochemistry, steps involved, prokaryotic DNA replication, Eukaryotic Replication).pptx

  • 1.
    Rashmi M G DNAreplication 1. Introduction to DNA replication 2. Methods of replication 3. Semi conservative replication 4. Meselson and Stahl experiment 5. Taylor's experiment 6. Replicon and origin of replication 7. DNA replication in prokaryotes 8. Biochemistry of DNA replication 9. Common enzymes involved in DNA replication 10. DNA polymerase 11. Components of DNA Polymerase III 12. Steps involved in prokaryotic DNA replication 13. DNA replication in eukaryotes 14. Unique aspects of eukaryotic DNA replication 15. Eukaryotic DNA Polymerases 16. Proof reading 17. Comparison between DNA replication in Prokaryotes and Eukaryotes
  • 2.
    Rashmi M G DNAreplication: • Transmission of chromosomal DNA from generations to generations • Achieved when chromosomal DNA is accurately replicated • Pairing AΞT, G=C-important role in replication • Providing two copies of the entire genome • For distribution into each daughter cells 2 strands of parental double helix of DNA separated Base sequence of each parental strand –serve as template Synthesis of new complementary strand 2 identical progeny double helices Parental double helix is half conserved (each parental strand (single) remaining intact) Semi conservative replication Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 6
  • 3.
    Rashmi M G Methodsof replication Conservative replication Whole original double helix acts as template for a new one One daughter molecule would consist of original parental DNA 2nd daughter had totally new DNA Dispersive replication Some parts of double helix are conserved and some parts are not Parental double strand helix, broken into double stranded DNA segments Synthesis of new double stranded DNA segment= same as conservative Semi conservative replication Parental double helix is half conserved, each parental single strand remaining intact Each parental strand acts as template
  • 4.
    Rashmi M G Source:Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 222
  • 5.
    Rashmi M G Meselsonand Stahl experiment Aim : To Demonstrate of semi conservative replication of DNA in E coli E coli cells Grown in Medium of Nitrogen source (15 N labeled NH4Cl- heavy isotopes of nitrogen) E coli cells culture Transferred to medium of nitrogen source (14 N labeled NH4Cl- light isotopes of nitrogen Allowed to grow continuously Samples were harvested at regular intervals DNA extraction and Isolation its buoyant density determined by centrifugation in CsCl density gradient Showed single band in density gradient
  • 6.
    Rashmi M G Source:Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.272,273
  • 7.
    Rashmi M G Result: Source:Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 224 Conclusion: DNA replication follows Semi conservative method
  • 8.
    Rashmi M G Taylor’sexperiment Aim: to suggest and conclude the semi conservative method of replication with the help of Vicia faba Root tips of Vicia faba ( broad beans) Chromosomes isolated and duplicated with thymidine labeled Observed in autoradiography Allowed to duplicate without labeled thymidine Again observed in autoradiography Conclusion: Proves the semi conservative replication of chromosome in broad bean Result: Source: Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.273
  • 9.
    Rashmi M G Repliconand origin of replication • DNA replication starts at particular site- origin of replication • Unit of DNA in which replication starts from an origin and proceeds bidirectionally/ unidirectionally to terminus site- replicon (may be linear / circular) Replicon Monorepliconic Bacterial genome Whole bacterial genome= a single replicon Contains single replication origin Multirepliconic Eukaryotic genome Eukaryotic cells has multiple replication origins on single chromosomes Origin of replication • Cis acting sequence • Fully methylated origin • Can initiate replication • In E coli, the origin of replication is oriC • It has 245 bp of DNA • Contains 2 short repeat motifs (9 nucleotides and 13 nucleotides • 9 Nucleotides repeat, 5 copies of which are dispersed throughout oriC- Binding site for a protein- Dna A • 13 Nucleotides repeat, located at one end of the oriC Sequence In prokaryotes, replicon- circular Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 227
  • 10.
    Rashmi M G Commonfeatures of replication origins: • Replication origins are unique DNA segments that contain multiple short repeated sequences • Three short repeat units are recognized by multimeric origin binding proteins. • These proteins play a key role in assembling DNA polymerase and other replication enzymes at the sites, where replication begins • Origin regions usually contain AT-rich stretch. This property facilitates unwinding of duplex DNA because less energy is required to melt A-T base pairs Replication fork In each replicon, replication is continuous from origin to terminus Accompanied by movement of replicating point- replication fork Replication may be, unidirectional or bidirectional Unidirectional replication Single replication fork in one direction Special types Chloroplasts, mitochondria and Plasmids Bidirectional replication 2 replication forks move in opposite directions Eukaryotes (common) Bacteria
  • 11.
    Rashmi M G Source:Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 229
  • 12.
    Rashmi M G DNAreplication in Prokaryotes (E coli)
  • 13.
    Rashmi M G DNAreplication in E coli: • Bidirectional replication • From a single origin –OriC • Replicon- circular with no free ends • Replication of DNA in E coli= Theta replication • 3 steps: • Initiation – recognition of the position on a DNA molecule where replication will begin • Elongation- events occurring at the replication fork, where parent polynucleotides are copied • Termination – occurs when parent molecule has been completely replicated
  • 14.
    Rashmi M G Biochemistryof DNA replication: In E coli >20 proteins take part in replication Proteins (Enzymes) Initiation • DnaA • Single stranded DNA Binding Protein (SSB) • DnaC • DnaG (Primase) Elongation • DNA polymerase • SSB • DNA gyrase • DNA ligase Termination • Terminus binding protein (Tus) • DNA Topoisomerase IV
  • 15.
    Rashmi M G DNAhelicase and primase • Opens up the duplex at the replication fork to provide a single stranded template • Primary replicative helicase- DnaB – binds to and moves on the lagging strand template in the 5’→3’ direction unwinding the duplex as it goes • Requires ATP hydrolysis SSB Protein • Inhibition of reannealing of separated strands • Bindings to both separated strands Primase • Synthesizes a short RNA primer (<15 nucleotides) to prime DNA chain elongation • Primosome- complex between primase and helicase, sometimes with accessory proteins Topoisomerase • Required to relieve the positive super coiling that arises from DNA unwinding • In E coli this role is typically fulfilled by DNA gyrase • In eukaryotes TopoIB DNA Polymerase • Catalyze the synthesis of DNA • Both prokaryotes and Eukaryotes contain multiple DNA dependent DNA polymerase • Termed as DNA Replicase • Performs DNA repair also Common enzymes and proteins involved:
  • 16.
    Rashmi M G DNApolymerase Template dependent DNA polymerase Template independent DNA polymerase DNA dependent DNA polymerase RNA dependent DNA polymerase (Reverse transcriptase)
  • 17.
    Rashmi M G InEubacteria, - E coli 5 types of DNA polymerases DNA polymerase I • Kornberg enzyme • Arthur Kornberg enzyme • Monomeric protein • Possess 3 enzymatic activities: • 5’3’ Polymerase activity • 5’3’ exonuclease activity • 3’5’ exonuclease activity • Primer removal function (with 5’3’ exonuclease activity) • Gap filling (5’3’ polymerase activity) and DNA repair DNA polymerase II • Monomeric protein • 5’3’ Polymerase activity • 3’5’ exonuclease activity • Alternative DNA repair polymerase • Can replicate DNA if the template is damaged • Low polymerizatio n rate DNA polymerase III • Primary enzyme • Multiprotein complex • (10 distinct polypeptides) • High polymerization rate • High Processivity DNA polymerase IV • Y family DNA polymerase • dinB • Do not contain 3’5’ exonuclease activity • Low catalytic efficiency • Low Processivity • Low fidelity • Involved in tranlesion synthesis • Replicate damaged DNA by bypassing damaged nucleotides DNA polymerase V • Y family DNA polymerase • Found in bacteria like E coli • Error prone polymerase • Involved in SOS response to extensive DNA damage
  • 18.
    Rashmi M G Source:Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 235
  • 19.
    Rashmi M G Componentsof DNA Polymerase III Catalytic core Dimerization component Processivity component Clamp loader α β θ τ Causes 2 catalytic core at replication fork to link together and form asymmetrical dimer Homodimer of β subunits (acts as sliding clamp) 5 subunits subassembly γ δ χ ψ δ϶ 5’3’ Polymerase activity 3’5’ exonuclease activity (Proof reading) To enhance proofreading activity of ϵ ϒ complex Loading of β clamp onto the DNA Facilitated by ϒ complex Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 241
  • 20.
    Rashmi M G ENZYME EXONUCLEASEACTIVITIES Subunits 3’5’ 5’3’ Function DNA polymerase I 1 YES YES DNA repair Gap filling Primer removal DNA polymerase II 1 YES NO DNA repair DNA polymerase III 10 YES NO Main replicating enzyme Major DNA polymerase involved in replication of bacterial genome
  • 21.
    Rashmi M G Stepsinvolved in prokaryotic DNA replication
  • 22.
    Rashmi M G DnaAprotein + oriC (9 mer sequence) =Initial complex (facilitates initial strand separation or melting) Occurs at oriC 13 mers Melting of 2 strands generates unpaired template strands Mediated by DnaB protein- Helicase Requires ATP and forms open complex 1 molecule= hexamer of identical subunit Clamp around each 2 single strand in open complex formed between DnaA and oriC Requires ATP and DnaC acts as helicase loader Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 234 Initiation step: DnaA protein- initiates replication in E coli at oriC
  • 23.
    Rashmi M G Helicase Movealong DNA duplex Utilize energy of ATP hydrolysis to separate strands SSB Protein Inhibits reannealing by binding to both separated strands Primase Synthesize short primer RNA complementary to both strands of DNA duplex Image showing the importance of SSB protein
  • 24.
    Rashmi M G Elongationreaction: Nucleophilic attack by 3’ hydroxyl group of primer on innermost phosphorus atom of deoxyribonucleoside triphosphate Phosphodiester bridge forms= release of pyrophosphate Catalytic metal ions (Mg2+ ) present in active site has important role Metal ions+ 3’OH = Reduce association between O and H leaves a nucleophilic 3’O- After catalysis pyrophosphate product stabilized through similar interaction with metal ions Hydrolysis by pyrophosphatase Metal ions + Triphosphate of incoming dNTP Neutralize their negative charge Helps to drive polymerization forward Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 232 Elongation step: DNA polymerase catalyze step by step addition of deoxyribonucleotide units to DNA chain
  • 25.
    Rashmi M G Source:Peter J Russell, iGenetics A molecular approach, 3rd edition, Page no.41 Source: Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.275
  • 26.
    Rashmi M G Ateach growing fork, 1.Leading strand: Synthesized continuously from single primer on leading strand template Growing fork Grows 5’3’ 2.Lagging strand: Complicated, DNA polymerase can add nucleotide only to 3’ end of primer / growing DNA strand Growing fork Leading strand growth Growing fork Leading strand growth Short pieces of DNA= Okazaki fragments – repeatedly synthesized on lagging strand template
  • 27.
    Rashmi M G Primase+helicase = Primosome Makes RNA primer DNA Polymerase III (synthesizing leading strand copy) extends RNA primer Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 237
  • 28.
    Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 331
  • 29.
    Rashmi M G Aseach Okazaki fragment formation completes, the RNA primer of previous fragment is removed by the 5’3’ exonuclease activity of DNA polymerase I This enzyme also fills in the gaps between the lagging strand fragments, which then are ligated together by DNA ligase Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 236
  • 30.
    Rashmi M G Terminationstep: • In bacterial genome, replication is bidirectional from single point • 2 replication fork should meet at a position diametrically opposite the origin of replication • Replication terminates at terminus region Terminus region: • Contains multiple copies of about 23 bp sequences called Ter (for terminus) • Each acting as recognition site for a sequence specific DNA binding protein called Tus (terminus utilization substance) Tus protein+ Ter sequence • Allows replication fork to pass if fork is moving ion one direction • Blocks progress if fork is moving in opposite direction around genome • Directionality is set by orientation of Tus protein on double helix • When approached from one direction- Tus blocks the passage of DnaB Helicase • When approached from other direction- DnaB –able to cross Tus protein Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 332 Completion of replication in circular chromosome, 2 new circles may be physically interlocked/ catenated Separated so that each daughter cell receives single dsDNA upon cell division Decatenation of interlocked circle- By Topoisomerase IV
  • 31.
    Rashmi M G Source:Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.274
  • 32.
    Rashmi M G DNAreplication in Eukaryotes
  • 33.
    Rashmi M G Tosummarize, Steps involved in DNA replication in Prokaryotes 1. DnaA protein+ oriC (9 mer)- Initial complex (ATP NEEDED) 2. DnaB protein- Helicase- for further melting 3. DnaC- Helicase loader- joins initial complex 4. SSB protein – Keeps unwound DNA strand in extended form 5. RNA primase- DnaG- For primer synthesis 6. DNA polymerase- Initiation of DNA polymerization 7. DNA polymerase I- Removal of primer 8. DNA polymerase II- DNA repair activity 9. DNA polymerase III- Main replicating enzyme- chain elongation, proof reading 10.DNA ligase- Joining of fragments (ATP NEEDED) The eukaryotic DNA replication is similar to prokaryotic DNA replication but differences lies in these following aspects, 1. Multiple replication origins in their chromosomes 2. More types of DNA polymerases with different functions 3. Linear DNA replication 4. Nucleosome assembly immediately after DNA replication 5. Different termination strategy
  • 34.
    Rashmi M G Uniqueaspects of eukaryotic DNA replication 1. Multiple replication origins • Eukaryotic genome= greater amount of DNA • Ex. Yeast, Drosophila • Replication takes more time • To facilitate the rapid synthesis of large quantities of DNA, Eukaryotic chromosomes contain multiple replication origins • Origins in yeast- Autonomously replication sequences • Eukaryotic replication origins- not only acts as sites of replication initiation, but also control the timing of DNA replication • These regulatory functions are carried out by a complex of more than 20 proteins, called Pre-replication complex (Pre-RC), which assembles at replication origins. Source: Klug cummins, Spencer, Palladino (2012), Concepts of Genetics, Pearson Publication, 10th Edition, Page no.282
  • 35.
    Rashmi M G 2.More types of DNA polymerases with different functions Eukaryotic cell contains, Many types of DNA polymerase Present both in nucleus and organelles (Mitochondria and chloroplast) 3 major types of DNA polymerase (present in nucleus for nuclear DNA replication): 3. DNA polymerase α 4. DNA Polymerase δ 5. DNA polymerase ϵ Enzyme Exonuclease activity 3’5’ 5’3’ Function DNA polymerase α NO NO Priming during replication DNA polymerase β NO NO Base excision repair DNA polymerase γ YES NO Mitochondrial DNA replication DNA polymerase δ YES NO Lagging and leading strand synthesis DNA polymerase ϵ YES NO - Major DNA polymerase involved in Replication of Eukaryotic genomes
  • 36.
    Rashmi M G EukaryoticDNA polymerases 1. DNA polymerase α • Unusual polymerase • Has both DNA polymerase and primase activity • 5’3’ DNA dependent DNA polymerase activity • 5’3’ DNA dependent RNA polymerase activity • Heterotetramer Primase activity: • Incorporates NMP- To synthesize short RNA primer (iRNA initiator RNA) • Complement – ssDNA template Polymerase activity: • Adds 20-30 NMP to 3’ end of iRNA (also DNA – iDNA) • No proofreading activity • (absence of 3’ 5’ exonuclease activity Function: • Initiates DNA synthesis on both leading and lagging strand • Polymerase α + initiation complex- at origin: • Synthesize short iRNA followed by 20-30 bases of iDNA • Then replaced by other DNA polymerase- extend the chain • This process is called- polymerase switching
  • 37.
    Rashmi M G 2.DNA Polymerase δ • Multi subunit nuclear enzyme • 4 subunits • Has 5’3’ polymerase activity • 3’5’ exonuclease activity • Requires associated 30kDa-protein (proliferating cell nuclear antigen PCNA) function as sliding clamp • For high Processivity • Requires clamp loader- replication factor C acts as clamp loader Function: Alone replicates leading and lagging strands of DNA 3. DNA polymerase ϵ • Multi subunit nuclear enzyme • 4 subunits • 5’3’ polymerase activity • 3’ 5’ exonuclease activity • High processive DNA synthesis with help of sliding clamp and clamp loader • Function: Not known precisely 4. DNA polymerase γ • Sole polymerase • Participate in mitochondrial DNA replication • 3 subunits • 5’3’ polymerase activity • 3’5’ exonuclease activity
  • 38.
    Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 329
  • 39.
    Rashmi M G 3.Nucleosome assembly immediately after DNA replication • Eukaryotic DNA is complexed to histone proteins in nucleosome structures that contribute stability and packing of the DNA molecule. • The disassembly and reassembly of nucleosomes on newly synthesized DNA probably takes place in replication, but the precise mechanism for these processes has not yet been determined • Before replication, a single DNA molecule is associated with histone proteins • After replication and nucleosome assembly, two DNA molecules are associated with histone proteins Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 247
  • 40.
    Rashmi M G 4.Termination step in Eukaryotic replication 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ RNase H removes the primer, up to the last ribonucleotide 5’ 3’ 3’ 5’ FEN1 removes that last ribonucleotide 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ DNA ligase links the two DNA fragments Helicase breaks the H- Bond between primer and template 5’ 3’ 3’ 5’ Removal of the primer 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ DNA ligase links the two DNA fragments Rnase H model The Flap model FEN1 cuts at the junction
  • 41.
    Rashmi M G Proofreading •Needed by mammals ( in large genomes) • Scanning the termini of nascent DNA chain for errors • Correcting them before continuing chain extension • Carried out by 5’3’ exonuclease activity that is built into DNA polymerases • When template- primer DNA has a terminal mismatch (an unpaired/ incorrectly paired base/ sequence of bases at 3’ of primer) • 3’5’ exonuclease activity of DNA polymerase I or II hips off the unpaired base/ bases • When appropriate base- paired terminus is produced • The 5’3’ polymerase activity of enzyme begins resynthesis by adding nucleotides to 3’ end of primer strand Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 339
  • 42.
  • 43.
    Rashmi M G Enzyme/Protein E coli Human Helicase DnaB MCM ssDNA binding protein SSB RPA (Replication protein A) Primase DnaG Dna polymeraseα/ Primase Replicase DNA polymerase III DNA polymerase ϵ / DNA polymerase δ Topoisomerase Gyrase Topo I, II Processivity component β clamp PCNA (Proliferating cell nuclear antigen) Clamp loader γ-complex RFC (Replication factor C) DNA replicating enzymes/ proteins from E coli and Human
  • 44.
    Rashmi M G Quantitativeparameters E coli Human DNA content, number of nucleotide pairs per cell 3.9✗ 106 About 109 Rate of replication fork progression per replication (per second) ~1000 ~100 Number of replication origins per cell 1 103 -104 Time required for complete genome replication ~42 minutes ~8hours Comparison of quantitative parameters of DNA replication in E coli and Human