OLM-16.1 DNA Replication.pptx

DNA REPLICATION Dr. David Rodda, PhD Learning Objectives 1. Describe and contrast the process of DNA replication in prokaryotes and eukaryotes. 2. Explain the functions of the enzymes and proteins required for both prokaryotic and eukaryotic DNA replication. 3. Analyze a DNA replication fork to id...

DNA REPLICATION Dr. David Rodda, PhD Learning Objectives 1. Describe and contrast the process of DNA replication in prokaryotes and eukaryotes. 2. Explain the functions of the enzymes and proteins required for both prokaryotic and eukaryotic DNA replication. 3. Analyze a DNA replication fork to identify the strands and indicate where proteins will act. 4. Describe the function of and classify nucleic acid polymerases. 5. Differentiate the activities of DNA polymerases and identify which polymerases have each activity. 6. Explain the functions of reverse transcriptases and telomerases. 7. Describe cDNAs and explain why they are useful in molecular biology. Principles of DNA Replication DNA Replication in Prokaryotes Topoisomerases DNA Replication in Eukaryotes 2 Special Polymerases • • Reverse Transcriptase Telomerase Summary PRINCIPLES OF DNA REPLICATION DNA Replication is Semiconservative ü û û DNA Polymerases • DNA-dependent DNA polymerase • Read the DNA template in the 3' to 5' direction • Synthesizes a complementary DNA strand in the 5' to 3' direction  Called a 5' to 3' DNA Polymerase activity  Nucleotides are added one at a time to the growing 3’ end A phosphodiester bond is created between the 3’ end and the new nucleotide  DNA Chain Elongation • Substrates: deoxynucleotide triphosphates (dNTPs) • Bond forms between 3'-OH of growing strand and first phosphate  A pre-existing 3’-OH is required • The second and third phosphates are released as inorganic pyrophosphate (PPi) DNA Polymerases Require a Primer DNA polymerases require an existing 3'-OH to which nucleotides are added Primase • DNA-dependent RNA polymerase • Synthesizes a short RNA primer (~10 nt) complimentary to the template • Provides the necessary 3'OH for the DNA polymerase (a) Not templates for DNA Polymerases (b)Good templates for DNA Polymerases • Nb. RNA polymerases, unlike DNA polymerases, do not require primers Other Important Proteins in Replication DNA Helicases • Enzymes that separate (unwind) the strands of dsDNA  Bind one strand, translocate along it & displace the other strand • Require energy from ATP to break the H-bonds • Create supercoils in front and behind the replication fork Single-Stranded DNA Binding Proteins (SSBs) • Protect single-stranded DNA from nuclease attack • Prevent reannealing Topoisomerases • Prevent formation of supercoils The Replication Fork In a diagram of a replication fork you should be able to identify all the strands and their directionality • In motion: moves into the double stranded parent DNA • Leading strand is synthesized 5' to 3' in the same direction as fork movement • Lagging strand is synthesized 5' to 3' in the opposite direction as fork movement   Can't be synthesized continuously Synthesized as small fragments called Okazaki Fragments which are later ligated Replication Origins • DNA Replication can only begin at specific sequences called Origins of Replication • From every origin two replication forks begin, migrating in opposite directions • Nb. One strand will be the leading strand for one fork, but the lagging strand for the other fork Visualization of bidirectional replication of a plasmid in E.coli Proofreading • DNA Polymerases make a mistake every 10,000-100,000 bp Proofreading • Removes incorrect, mispaired nucleotides introduced by DNA polymerase errors • A 3'-5' exonuclease activity   Back-up and cut off the incorrect nucleotide Found in many, but not all DNA polymerases • Improves fidelity of replication by 100 1000 fold Principles of DNA Replication DNA Replication in Prokaryotes Topoisomerases DNA Replication in Eukaryotes 2 Special Polymerases • • Reverse Transcriptase Telomerase Summary DNA REPLICATION IN PROKARYOTES Initiation of Prokaryotic DNA Replication • One replication origin • Initiator protein   Binds the origin Induces initial melting • Helicase    Binds to initiator Separates the strands of double-stranded DNA Creates 2 replication forks and 2 ssDNA templates Primers on Leading and Lagging Strands • Primase synthesizes RNA primers on each new chain   1 primer on leading strand Multiple primers on lagging strand to begin each Okazaki fragment primers 3’ 5’ 5’ 3’ Coordinated Synthesis of DNA Strands DNA Polymerase III • Synthesizes the leading and lagging strands • Activities:   5'-3' DNA Polymerase (DNA synthesis) 3'-5' Exonuclease (Proofreading) Primer Removal • When DNA Polymerase III on the lagging strand hits the preceding RNA primer it releases from the DNA strand • A "nick" separates the completed Okazaki fragment and the RNA primer  Nick = adjacent nucleotides not connected by a phosphodiester bond DNA polymerase I • Binds to the nick and removes the RNA  5' to 3' Exonuclease Activity • Simultaneously replaces the RNA by synthesizing DNA  5' to 3' Polymerase Activity • And proofreads the new DNA for errors  3' to 5' Exonuclease Activity DNA Ligase • Closes the final nick between the DNA fragments Primer Removal by DNA Polymerase I Both the 5'-3' Exonuclease and the 5'-3' Polymerase activities are at work DNA Ligase DNA Ligase • Catalyzes the formation of a phosphodiester bond between:   5'-Phosphate on DNA chain synthesized by DNA polymerase III 3'-OH on DNA chain synthesized by DNA polymerase I Comparing the E. Coli DNA Polymerases DNA Pol III DNA Pol I 5'-3' Polymerase (DNA Synthesis) 3'-5' Exonuclease (Proofreading) 5'-3' Exonuclease (Primer Removal) Polymerization Rate Yes Yes Yes Yes No Yes High Low Processivity High Low Replication Animation Principles of DNA Replication DNA Replication in Prokaryotes Topoisomerases DNA Replication in Eukaryotes 2 Special Polymerases • • Reverse Transcriptase Telomerase Summary TOPOISOMERASES DNA Supercoiling • As DNA strands are separated the dsDNA rotates  Creates torsional stress • Supercoils form   Relieve torsional stress Supercoils can be: • • Positive due to overwinding Negative due to underwinding • Supercoils interfere with DNA replication   Must be removed for replication to proceed Otherwise, growth stops & eventual cell death Topoisomerases • Enzymes that remove supercoils • 2 Families:  Type I • •  Type II • • During replication, a topoisomerase sits in front of the replication fork, removing positive supercoils and acting like a swivel Cut one strand (creating a nick) Don't require energy Cut both strands Require energy from ATP • Drug targets   Topoisomerase inhibitors block unwanted cell growth Examples: • • Bacterial infections Cancer DNA Gyrase • A Type II topoisomerase • Introduces negative supercoils   Using energy from ATP Neutralizes the positive supercoils introduced by strand unwinding during DNA replication Bacterial Topoisomerase Inhibitors Bacterial Type II topoisomerase (Gyrase) inhibitors • Antibiotics • Example:  Ciprofloxacin (aka Cipro) Selective Toxicity • Antimicrobial drugs harm the target microbe but not the human host • Bacterial type II topoisomerase inhibitors have a low affinity for eukaryotic type II topoisomerases so are non-toxic to humans Eukaryotic Topoisomerase Inhibitors Eukaryotic topoisomerase inhibitors • Chemotherapeutics • Examples: • Type I topoisomerase inhibitors  Irinotecan, topotecan • Type II topoisomerase inhibitors  doxorubicin, etoposide Principles of DNA Replication DNA Replication in Prokaryotes Topoisomerases DNA Replication in Eukaryotes 2 Special Polymerases • • Reverse Transcriptase Telomerase Summary DNA REPLICATION IN EUKARYOTES Major Differences 1. Cell Cycle In eukaryotes, replication only occurs during S Phase Cell cycle checkpoints control transition through the stages   • G1/S Checkpoint controls entry into S Phase Replication must occur only once per cell cycle Replication will only occur if there is no DNA damage Major Differences 2. Very long, linear chromosomes vs. shorter, circular chromosomes 3. Multiple origins of replication vs. a single origin  Every 50,000 – 100,000 bp 4. Different enzymes/proteins carry out the functions Eukaryotic Replication Origins • Eukaryotic replication origins are much larger and more complex than bacterial origins Key proteins: Origin of Replication protein Complex (ORC) • Binds to origins of replication • Initiation of replication Minichromosome Maintenance Complex (MCM) • Helicase • A hexameric complex of proteins MCM2 to MCM7 • Tightly regulated, active only in S-phase. Eukaryotic DNA Polymerases DNA Polymerase α • A complex containing i. ii. Primase DNA Polymerase • Synthesizes a short RNA primer (~10nt) then switches to DNA synthesis (20-30nt) • No proofreading DNA Polymerase δ • The primary highly-processive polymerase • Synthesizes the leading and lagging strand • Uses a sliding clamp called PCNA DNA Polymerase ε • An alternative highly-processive polymerase • Also involved in leading and lagging strand synthesis and DNA repair Eukaryotic Replication Primer Removal • Primers are NOT removed by a 5'-3' exonuclease activity of a DNA polymerase  There is no eukaryotic equivalent of DNA polymerase I • DNA polymerase δ or ε displace the RNA primer as a “flap”, and continue synthesizing DNA FEN1 (Flap Endonuclease) • Endonuclease that remove the RNA primer “flap” • Alternatively RNaseH can remove RNA primer DNA Ligase • Closes the final nick between the DNA strands after the RNA primer has been removed The Eukaryotic Replication Fork 5’ 3’ MCM Helicase 3’ 5’ (activity) 3’ 5’ 5’ 3’ Principles of DNA Replication DNA Replication in Prokaryotes Topoisomerases DNA Replication in Eukaryotes 2 Special Polymerases • • Reverse Transcriptase Telomerase Summary 2 SPECIAL POLYMERASES Reverse Transcriptase • RNA-dependent DNA polymerase • Reads an RNA template 3'-5' and synthesizes a complementary DNA strand 5'-3' • No proofreading • Reverse transcriptases are carried by retroviruses Generation of cDNA cDNA (complementary DNA) • A DNA copy of a mRNA • Generated using reverse transcriptase • cDNAs contain the entire coding sequence for a protein, but no introns  Introns are removed during mRNA splicing • Useful in many applications  Eg. Hybridization assays, recombinant proteins, transgenic organisms etc. Telomeres • Specialized repetitive DNA sequences at ends of chromosomes  6bp tandem repeats • Protect the ends of chromosomes • Permit complete replication of genetic information • Essential regulators of chromosomal integrity during cell division The End Replication Problem • Synthesis of the new strands can't extend completely to the 5'-ends  There is no 3'-OH available • The ends of the chromosomes get shorter after every replication • Telomeric repeats act like "buffers"  Telomeres shorten instead of important genetic information in the chromosome Cellular Senescence Cellular Senescence (G0 phase) • Cells stop dividing but remain metabolically active • Triggered by: i. ii. iii. Extensive telomere shortening (replicative senescence) DNA damage Oncogene signaling Replicative Senescence • Somatic cells can’t divide indefinitely • When telomeres become too short the cell will enter senescence • Hayflick Limit: maximum number of divisions a cell can undergo before entering senescence  Usually 40-60 cell divisions Telomerase • RNA-dependent DNA polymerase • A ribonucleoprotein  A complex of protein and RNA • Uses its own RNA as a template • Repeatedly adds the repeat sequence to the 3' ends of chromosomes   Only one strand is synthesized by telomerase The other strand is synthesized by standard DNA replication • Telomerase is active in germ cells, and the inner cell mass cells/epiblast of blastocyst-stage embryos • Normally not active in adult somatic cells, but reactivated in cancer Principles of DNA Replication DNA Replication in Prokaryotes Topoisomerases DNA Replication in Eukaryotes 2 Special Polymerases • • Reverse Transcriptase Telomerase Summary SUMMARY Summary 1 1. DNA replication is semi-conservative. One strand is used as a template for synthesis of the other strand. This allows for a very low error-rate. 2. Polymerases are classified using this system: 3. All nucleic acid polymerases (both DNA and RNA) synthesize their nucleic acids in the 5’ to 3’ direction while reading their templates in the 3’ to 5’ direction. This is called a 5’ to 3’ polymerase activity. 4. Nucleotides are added to a growing DNA strand as nucleotide triphosphates, creating a phosphodiester bond between the 5’ phosphate on the new nucleotide and the 3’ hydroxyl on the existing strand. 5. DNA polymerases, unlike RNA polymerases, require a pre-existing 3’-OH to begin synthesis. The 3’OH is provided by a primer. 6. Both strands of the parent molecule are used as templates for synthesis. One strand becomes the leading strand, the other the lagging strand. 7. Leading strands are synthesized continuously, lagging strands are synthesized discontinuously in segments called Okazaki fragments. 1. From the given structure, identify: -5' A. B. C. 3'5'- 3' Direction of fork movement Leading strand template Lagging strand template 2. Draw the leading and lagging strands, indicating direction of synthesis 3. Point out where each of the proteins on the previous slide would act at the fork Summary 2 8. Most DNA polymerases have 3’ to 5’ exonuclease activity which is used to correct errors – called ‘Proofreading’. 9. The requirements for DNA synthesis are listed on the next slide. 10. In Prokaryotes, DNA Polymerase III is the major polymerase that synthesizes the leading and lagging strands. 11.DNA Polymerase I removes the RNA primers with its 5’ to 3’ exonuclease activity and replaces them with DNA using its 5’ to 3’ polymerase activity. 12.DNA Ligase creates phosphodiester bonds at ‘nicks’ – for example between Okazaki fragments on the lagging strand. 13.DNA supercoiling occurs when strands separate Eg. during replication and transcription. Supercoils interfere with replication and transcription so must be resolved for these processes to continue. 14.DNA topoisomerases remove supercoils from DNA. DNA gyrase is a special Type II topoisomerase found in bacteria that introduces negative supercoils to cancel out the positive supercoils created in replication. 15.Gyrase inhibitors are used as antibiotics. Eukaryotic topoisomerase inhibitors are used as chemotherapeutics. Requirements for DNA Replication 1. 2. 3. 4. Template DNA Primer All four dNTPs Proteins: 1. 2. 3. 4. 5. 6. 7. 8. Initiator Proteins DNA Polymerases Topoisomerase Helicase Primase Single-Stranded DNA Binding Proteins Nucleases Ligase Summary 3 16.Eukaryotic replication occurs only during S-phase and is carefully controlled by cell cycle checkpoints. 17.In eukaryotes, long, linear chromosomes are replicated from multiple origins of replication. 18.The requirements for eukaryotic replication are the same as for prokaryotic replication, except that different enzymes carry out the processes (see next slide). 19.Reverse transcriptases are DNA polymerases that read an RNA template. They are naturally occurring in retroviruses and are used to generate cDNA. 20.cDNA is a DNA copy of the mature mRNA sequence. It is useful if a number of molecular biology applications because it contains the coding sequence without introns. 21.Linear chromosomes can’t be replicated completely because they have ends. Thus chromosomes shorten with each cell division. 22.Telomerase extends the ends of chromosomes in germ cells and in the inner cell mass of blastocyst stage embryos. Telomerase is inappropriately activated in cancer. Summary of Prokaryotic and Eukaryotic Replication Proteins Function Prokaryotic Eukaryotic Initiation Initiator proteins ORC Unwinding Helicase MCM Helicase Remove Supercoils Topoisomerase & Gyrase Topoisomerases Priming Primase DNA Pol α Primary DNA Synthesis Removing Primers DNA Pol III DNA Pol δ and ε DNA Pol I FEN1 Gap Filling DNA Pol I DNA Pol δ and ε Sealing The Nick DNA Ligase DNA Ligase DNA Polymerases in Replication and 