Current LectureMCB 250 1st Edition Lecture 11Outline of Current Lecture 1. Replication fork2. Helicase- DnaB3. SSBP4. Primase5. E. Coli DNA POL III6. DNA replication7. Beta Subunit8. Protein9. RNAseCurrent Lecture1. Replication Fork- DNA polymerase synthesizes DNA starting from a primer that supplies free 3’OH group. Synthesizes DNA 5’ to 3’. Have free 3’OH and incoming dntp which energy is supplied by triphosphate, then dntp will be incorporated depending on the base pair. Synthesize DNA 5’-3’ but the template runs in the other direction!- Replication fork: machinery which moves down the DNA and synthesizes both strands of DNA at the same time- Two strands run antiparallel and can only synthesize DNA 5’ to 3’. DNA replication is SEMI-CONTINUOUS: if one strand is synthesized continuously in the direction of the replication fork (leading strand) 5’-3’, but template is 3’-5’. The other strand is synthesized discontinuously. Made by synthesizing primers 5’-3’ and continuing synthesizing 5’-3’ and moves down direction of the fork movement. (lagging strand runs 5’-3’)- Replisome (synthesizes both strands at the same time). Lagging strand synthesized with Okazaki fragments and leading strand is synthesized continuously- Primers are made of RNA. Okazaki fragments primer is removed and information is still in, leaving a nick, which DNA ligase will fulfill. - ReplisomeSynthesizes one strand 5’ to 3’ and other strand Okazaki Fragment2. Helicase- DnaBThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Hexameric protein (6 copies of the same subunit). Wraps completely around the LAGGING strand. It travels by using ATP to unwind the helix and travels 5’ to 3’ down the lagging strand template to unwind the helix. Driving force of movement of replication fork down the DNA. It requires another protein (loading) onto the DNA. - Helicase is using ATP to travel down the lagging strand 5’ to 3’ to unwind the DNA. ENERGY DEPENDENT PHENEMONON. 3. Single Stranded Binding Proteins- Present throughout the cell. Whenever there is ssDNA, SSB will recognize it and bind to it and prevent it from folding up on itself or re-annealing.- SSB is highly cooperative: if you have ssDNA SSB doesn’t randomly bind. Once it binds, the protein is much more likely to polymerize along the ssDNA rather in a random spot. - Not just DNA replication, wherever there is ssDNA4. Primase- Recruited by helicase- Recognizes DNA sequence and lays down 10-12 nucleotide primer synthesizing it 5’-3’. The primer is RNA!!!- RNA primer based on DNA template and then it dissociates. It will fall apart- Every 1-2 kb on the lagging strand, helicase will recruit a primase and lay down a primer and then dissociate- Why RNA as a primer?o Leftover from “RNA world”: life begins as RNA- Protein(ribosomes made of RNA)-DNAo FIDELITY: DNA polymerase has to have a primer to start with. DNA polymerase cannot synthesize DNA ‘de novo’ from new. Need a primer to synthesizeo Laying down the first base there is no structure, it is difficult to get the geometry of that. o The cell uses RNA and may mess it up, but 10-12 bases down with a stable structure, the DNA polymerase will extend the rest. Then the RNA is marked and will be removed and filled with polymerase starting with 3’OH on the other side. o Don’t care if fidelity is quite right, delete, and filled with a high fidelity DNA5. E. Coli DNA POL III- Complex structure: 14 subunits- Core polymerase: machine doing DNA synthesis- All DNA polymerases have a similar “hand”- Travels down template strand and puts complementary bp- Enzyme: lower the activation energy required for the reaction to take place- Take the 2 substrates and put them in a conformation and control the chemistry so the reaction takes place- Incoming nucleotide if it fits appropriately with the template base, then the trinucleotide in the enzyme will be reacted with 2 divalent cations (metals). One metal ion interacts with phosphate groups, other helps extract proton from 3’ OH group. Oncethe proton is extracted and phosphate group is bent to an unstable confirmation, then the OH group will attack the phosphate and create a new phosphodiester bond. CLICKER: If next is T, how do all enzymes find dntpThere is a certain conc of all dNTP in the cell, there is no way to figure out what’s right unless you sample them. Always having come in active cell. Only if it’s right and fits in the complex, then the reaction will take place. Diffusion is very fast. There is just sampling of the dNTP’s until appropriate substrate6. DNA replication- Mistake = mutation- Can be corrected- Pol III can fix misincorporations but there are repair mechanisms that can repair mistakes it makes.- Pol III makes mistake within 1 in amillion. Overall is 1 in a billion due to repair machinery- Mechanism; if the correct base is not there, then it cannot have a reaction for it to occur- Concentration of ATP is 1000x more than dATP in the cell.- How does DNA pol III keep from incorporating RNA than DNA? The difference is the 2’OHgroup. DNA polymerase cannot fit 2-‘OH it will sterically hinder a NTP from getting into the active site and fitting appropriately. But, it will sample atp more often than datp. - Pol III has an ability to repair misincoproated base- Structure of the DNA is messed up if the base is not complementary and doesn’t get put together. The OH group wont be in the right place. The enzyme can recognize that and moves the DNA to another site in the enzyme and there is a 3’ to 5’ EXONUCLEASE activity that can remove the misincoporated base. Then move back to active site to be fixed- Between the 3 things, there are very rare misincoporations7. Beta subunit: Clamp- Sliding clamp: dimeric protein that has a big hole in the middle. Can wrap completely around dsDNA. It does not bind DNA, it slides along the DNA. It tethers the PolIII core to the DNA: processivity: makes the pol III PROCESSIVE.- It doesn’t interact with the DNA, plenty of water molecules in between- Pol III is interactive with the clamp and travels down the DNA with the clamp holding it in place- To make it processive, it holds the pol to the DNA so it wont let go. Without the clamp, pol III will bind and synthesize and fall off