Lecture #20 : DNA Replication- I Recommended reading: Chapter 11 – DNA Replication 1 Today’s lecture content: ① Universal principles of DNA replication DNA replication is semiconservative DNA synthesis needs priming DNA replication is polar (5’ to 3’): leading and lagging strands ① DNA replication in E. coli DNA polymerase activity: error rate and processivity Other activities required at the replication fork 2 ① Universal Principles of DNA Replication 3 ① Universal Principles of DNA Replication Replication is semiconservative • The complementary base pairing of the DNA helix suggested that each strand could act as a template for the synthesis of its complementary strand 4 ① Universal Principles of DNA Replication • What is the mechanism of DNA replication: • • • Conservative? Semi-Conservative? Dispersive? Meselson and Stahl experiments (1958) 5 ① Universal Principles of DNA Replication • Meselson and Stahl grew E. coli in growth medium containing ammonium 15NH4 (heavy Nitrogen Isotope) as a source of Nitrogen • They then switched to 14NH4 (light Nitrogen Isotope) • DNA with heavy or light Nitrogen sediment differently in a Cesium Chloride density gradient 6 ① Universal Principles of DNA Replication • Meselson and Stahl grew E. coli in growth medium containing ammonium 15NH4 (heavy Nitrogen Isotope) as a source of Nitrogen • They then switched to 14NH4 (light Nitrogen Isotope) • DNA with heavy or light Nitrogen sediment differently in a Cesium Chloride density gradient • In addition, they used either: • Native conditions where DNA was double stranded • or Alkaline, or denatured conditions, where DNA strands were separated 7 ① Universal Principles of DNA Replication 8 9 ① Universal Principles of DNA Replication 10 ① Universal Principles of DNA Replication • DNA replication is catalyzed by DNA polymerases, which have two properties with major implications for DNA replication: • They cannot initiate DNA synthesis de novo and require a “primer” with a free 3′-OH group at the 3’ end • They can extend DNA only in the 5′→3′ direction 11 ① Universal Principles of DNA Replication DNA Synthesis needs priming • Problem 1: They cannot initiate DNA synthesis de novo and require a “primer” with a free 3′-OH group at the end Solution: DNA synthesis is primed by an RNA! 12 ① Universal Principles of DNA Replication DNA replication is polar (5’ to 3’) • Problem 2: DNA polymerases can extend DNA only in the 5’ to 3’ direction but the two parental strands are anti-parallel Solution: Replication is semidiscontinuous: • The 3’-5’ strand (Crick strand) serves as template for the leading strand synthesis • The 5’-3’ strand (Watson strand) serves as template for the lagging strand synthesis 13 The leading strand can be extended continuously, with the polymerase moving from the 5’ terminus to the 3’ terminus in the same direction as fork movement. The lagging strand synthesis is discontinuous with short fragments (called Okazaki fragments) synthesized by the polymerase moving opposite to the direction of fork movement. The Okazaki fragments are then stitched together by the enzyme DNA ligase. Reiji & Tuneko Okazaki 14 Today’s lecture content: Universal principles of DNA replication DNA replication is semiconservative DNA synthesis needs priming DNA replication is polar (5’ to 3’): leading and lagging strands ② DNA replication in E. coli DNA polymerase activity: error rate and processivity Other activities required at the replication fork 15 ② DNA replication in E. coli • E. coli has been a crucial model system for dissecting DNA replication • It is a simpler model with a well defined origin and termination of replication sites, as well as replication forks moving in opposite direction 16 17 ② DNA replication in E. coli DNA polymerase activity • The E. coli DNA pol I is the most well studied DNA polymerase: 18 ② DNA replication in E. coli DNA polymerase activity • DNA pol I extends the DNA in the 5′→3′ direction incorporating a dNMP complementary to the template strand (A-T, G-C): 19 ② DNA replication in E. coli DNA polymerase activity • In addition to its DNA polymerase activity, the E. coli DNA pol I also has two exonuclease activities: • 3' →5' exonuclease activity that digests the DNA strand from the 3' terminus • 5' →3' exonuclease activity that digests the DNA strand from the 5' terminus 20 ② DNA replication in E. coli DNA polymerase activity • The 3' →5' exonuclease activity of DNA pol I is responsible for removing mis-incorporated dNMPs • This activity is called proofreading • DNA pol I has an error rate of 10-4 or 10-5, but this error rate is improved 102- to 103-fold by 3' →5' exonuclease proofreading activity. 21 22 ② DNA replication in E. coli DNA polymerase activity • The 5' →3' exonuclease activity of DNA pol I is responsible for repairing nicks and gaps between Okazaki fragments • This activity is called nick translation 23 At the gap between lagging strand fragments, DNA Pol I degrades the RNA primer in the 5'→3' direction, releasing NMPs, and simultaneously extends the 3' terminus with dNTPs in the same direction. The net result is movement of the nick in the 5'→3' direction along the DNA until all RNA is removed. DNA ligase can then seal the fragments (not shown here). 24 ② DNA replication in E. coli DNA polymerase activity • DNA Pol I is actually not the DNA polymerase responsible for the E. coli chromosome replication 25 ② DNA replication in E. coli DNA polymerase activity • DNA Pol III is the DNA polymerase responsible for the E. coli chromosome replication • The Pol III core is a heterotrimer that contains one each of: • α subunit, containing the DNA polymerase activity • ε subunit, containing the proofreading 3' →5' exonuclease activity • θ subunit, of unknown function 26 ② DNA replication in E. coli DNA polymerase activity • The Pol III core has a processivity of 1 to 10 nucleotides and synthesizes ~ 10 nucleotides per second • However, the replication of the E. coli chromosome occurs at a rate of 1000 bp per second! • During replication, Pol III associates with the β sliding clamp which dramatically increases its processivity and rate of DNA polymerization 27 ② DNA replication in E. coli DNA polymerase activity • The β sliding clamp forms a “doughnut” that completely surrounds the double-stranded DNA and thus keep the DNA Pol III associated with its DNA template 28 ② DNA replication in E. coli DNA polymerase activity • The β sliding clamp is loaded onto the primed DNA template by the γ complex clamp loader 29 ② DNA replication in E. coli DNA polymerase activity • The β sliding clamp increases the speed and processivity of DNA Pol III 30 ② DNA replication in E. coli DNA polymerase activity • For example, with a preloaded Pol III core and β sliding clamp, the phage ΦX174 DNA is synthesized in 11 sec! 31 ② DNA replication in E. coli Other activities required at the replication fork • Apart from the DNA polymerase activity, many other activities are required at the replication fork: • β sliding clamp and γ complex clamp loader for processivity 32 ② DNA replication in E. coli Other activities required at the replication fork • Apart from the DNA polymerase activity, many other activities are required at the replication fork: • DNA B 5’- 3’ helicase required for unwinding the DNA 33 ② DNA replication in E. coli Other activities required at the replication fork • Apart from the DNA polymerase activity, many other activities are required at the replication fork: • DNA topoisomerases required for relieving the positive supercoiling of the DNA ahead of the helicase 34 ② DNA replication in E. coli Other activities required at the replication fork • Apart from the DNA polymerase activity, many other activities are required at the replication fork: • Okazaki fragment priming and sealing 35 ② DNA replication in E. coli Other activities required at the replication fork • Apart from the DNA polymerase activity, many other activities are required at the replication fork: • Okazaki fragment priming and sealing 36 ② DNA replication in E. coli Other activities required at the replication fork • Apart from the DNA polymerase activity, many other activities are required at the replication fork: • Single strand protection 37 38 Next Lecture: #21 – DNA Replication - II On Monday, March 11th Recommended reading: Chapter 11: DNA replication 39