3400_L6_2024_DNA_replication_1.pdf

6. DNA Replication (1): Introduction Stanford News, 2007 Chapter 11, 363–402 (NOTE — unless otherwise indicated, chapter / page / figure numbers refer to the recommended text:...

6. DNA Replication (1): Introduction Stanford News, 2007 Chapter 11, 363–402 (NOTE — unless otherwise indicated, chapter / page / figure numbers refer to the recommended text: Cox et al.: Molecular Biology (Principles and Practice), 2nd Edition (2015) Rules of DNA replication 1. Uses deoxynucleoside 5’-triphosphates (dNTPs) to form the phosphodiester bond. Arthur Kornberg (left) (1987-2007) (dNTPs: dATP, dCTP, dGTP, dTTP) (not NTPs, not dNMPs, and not dNDPs). 1959 Nobel Prize 2. Semi-conservative replication of the two DNA strands. (DNA replication) See “HOW WE KNOW” 3. Usually starts at a specific sequence (replication origin), may be uni- or bi-directional. Pp. 406-407 Usually single origin for bacteria and viruses, multiple origins in eukaryotes. 4. Replication starts with production of a short RNA primer by primase enzyme. 5. Proceeds by the addition of dNTPs in the 5’ to 3’ direction (for both strands). Relatively continuous on the leading strand and discontinuous on the lagging strand. 6. Several different DNA polymerases are involved in different aspects of DNA replication. 7. Generally controlled at the point of initiation on a chromosome or genome. 8. Different genomes within a single cell may employ different mechanisms. BIOC3400 Archibald—L6 1  Elongation of a DNA chain DNA polymerase activity requires a single unpaired strand to act as template, and a primer strand to provide a free hydroxyl group at the 3′ end (3’-OH), to which a new dNTP unit is added. Each incoming dNTP is selected, in part, by base pairing to the opposing nucleotide in the template strand. The reaction product has a new free 3′ hydroxyl (3’-OH), allowing the addition of another nucleotide. The incoming nucleotide must be a dNTP with a 5’-triphosphate (not dNMP, dNDP, 3’-dNTP. WHY NOT? See next slide…) [Want to radioactively label newly synthesized DNA? Use [α-32P]dNTP. Further discussion in future lectures] BIOC3400 Archibald—L6 See also Fig 11-5 in Cox et al. 2  Why dNTP? Thermodynamics is important… Addition of 1 dNMP: ∆Go = +25 kJ/mol (phosphodiester bond) (reaction thus cannot occur by itself) dNTP à dNMP + PPi ∆Go = -31 kJ/mol (generation of PPi) PPi à 2 Pi ∆Go = -33 kJ/mol (hydrolysis of PPi) NET ∆Go = -39 kJ/mol (a negative number, reaction thus occurs efficiently) Generation and hydrolysis of PPi drives the reaction. NOTE: DNA polymerase can also perform pyrophosphorolysis: Pyrophosphate (PPi) using PPi, it removes dNMPs from primer strand, produces dNTPs Pyrophosphatase Take-home message: under cellular conditions, DNA replication is largely an irreversible process due to the elimination of PPi by O Pi + Pi replication at the the enzyme pyrophosphatase. ↳ He A forces direct BIOC3400 Archibald—L6 See also Fig 11-5 in Cox et al. 3  Semi-conservative DNA replication Parental double-stranded DNA (antiparallel strands, blue) Two double-stranded daughter DNAs Each double-stranded Daughter DNA has a parental strand (blue) and a daughter strand (red) How was this confirmed? The Meselson-Stahl experiment (1958) https://www.ibiology.org/genetics-and-gene-regulation/experiment-meselson-and-stahl/ BIOC3400 Archibald—L6 4  HYPOTHESIS – Three proposed models for DNA replication (Watson & Crick) After considering the models (hypotheses), Meselson and Stahl devised an experimental approach to distinguish the old parental DNA (blue) from the new daughter DNA (red): Use a heavy isotope (15N) to label old DNA and standard light isotope (14N) to label new DNA. Track the fate of ‘light’ and ‘heavy’ DNA as DNA is replicated in growing E. coli cultures using cesium chloride (CsCl) density gradient centrifugation. BIOC3400 Archibald—L6 5  EXPERIMENT – Track ‘heavy’ and ‘light’ DNA in E. coli Grow E. coli for multiple generations in medium containing 15NH4Cl as sole nitrogen source. All DNA ends up being uniformly labeled with 15N. Transfer 15N-labeled cells to unlabeled medium containing 14NH4Cl, allow cells to grow, and take samples every 20 minutes (i.e., E. coli generation time) Extract DNA from samples, mix DNA with CsCl solution, centrifuge DNA-CsCl solution at high speed, use ultraviolet light to visualize DNA fractions on the resulting CsCl gradient. This can be done with double-stranded (native) or single-stranded (alkaline-treated) DNA. Proof-of-principle (control) 50/50 mixture of [14N]DNA and [15N]DNA + CsCl salt solution BIOC3400 Archibald—L6 6  TESTING THE HYPOTHESES – Track ‘heavy’ and ‘light’ DNA in E. coli over time Native = double-stranded Alkaline = single-stranded 1st Edition CONCLUSION – semiconservative DNA replication See 11-c, d and e in 2nd Edition The more E. coli generations one examines, the ‘lighter’ the DNA becomes BIOC3400 Archibald—L6 7