Bisc320 Week 5 (Lectures 1 & 2)-Arthur Kornberg isolated protein enzyme DNA Polymerase I. Found that it could synthesized new strands of DNA, and that dNTP’s are require precursors of DNA(required in triphosphate form), and that a template of DNA was also necessary. Using templates with different base compositions (e.g. high A:T vs G:C or vice-versa), heshowed that the newly synthesized DNA molecules had a similar base composition by varying composition of the DNA template and introducing an A rich or G rich template. In the case of A-rich templates, molecule being produced would be T riched. Showed thatcomposition of the template determined the composition of the newly synthesized DNA. -Enzymatic Synthesis of DNA by DNA Pol1:SS region with the 3’OH is highlighted in red. Covalent bond forms between 3’OH on theT and the phosphate on the incoming dATP. Cleavage of pyrophosphate(high energy molecule), then cleavage by pyrophosphotase of the pyrophosphate drives the reaction b/c of the high energy associated with it, and because by cleaving the pyrophosphate, we have eliminated the product of the reaction (LeChattelier shift). The first cleavage provides high energy as well.-DNA Synthesis Requirements:Primer:template junction(ssDNA Template, and its complimentarily annealed primer witha 3’OH), dNTP’s, and DNA Polymerase. The alpha-beta is the bond that is cleaved. Growth/replication always occurs from 5’->3’, so 3’OH are the growing ends. All you need is enough complementary base pairs near the 3’ end to hold the strands together for replication to occur. -Mechanism of DNA synthesis: Correct base-pairing allows the reaction to take place by properly positioning the reactive groups in the polymerase active site.Hydrolysis of pyrophosphate drives the reaction.5’->3’. Phosphodiester bond is formed in an Sn2 reaction – hydroxyl groupat the 3’ end of the primerstrand attacks the alpha-phosphoryl group of the incoming dNTP. The leaving group = pyrophosphate. The dNTP that base-pairs with the template strand is highly favored for addition to the primer strand.-DNA Polymerase Right Hand shape: The structure will only close on the correct base-pair and catalyze the reaction. It allows the enzyme to hold on to the DNA, to repeat the catalytic cycle. Metal ions are used to shield the negative charges of the dNTPs and to activate the reactive 3’OH.It holds the DNA in the palm, unwinding it and placing the template inthe proper structure and orientation. Put metals in the right pockets to pull things in the right direction, making the OH more reactive. So the active site ispulling H off the OH, making it highly reactive so it will attack the phosphate bond on the dNTP. -Use a single active site to catalyze the addition of any of the four DNTP’s – has this catalytic flexibility by exploiting nearly identical geometry of the A:T and G:C base pairs. If the correct base pair forms, the 3’OH and alpha phosphate are in optimum position for catalysis to occur. If not, then won’t happen because too slow. DNA pol can also distinguish rNTP’s because there is steric exclusion of rNTPS from the DNA polymerase active site b/c 2’OH on the nucleotide interferes with the nucleotide-binding pocket(which is too small).-The palm region binds two divalent metal ions that alter the chemical environment around the correctly base-paired dNTP and the 3’-OH for its hydrogen. This generates a 3’O- that is primed for the nucleophilic attack of the alpha-phosphate of the incoming DNTP. The second metal ion coordinates the negative charges of the other phosphates of the dNTP and stabilizes the pyrophsphate produced by joining the primer and incoming nucleotide. Overall, the metal ions: pull the H off of the 3’OH, deshield the phosphate backbone, and stabilize the pyrophosphate productSummary of mechanism: Incoming nucleotide base pairs with chain. The “fingers” close around the base-paired dNTP. This confomation of the enzmye places the critical catalyticmetal ions in a position to catalyze formation of the next phosphodiester bond. Attachment of the nucleotide to the primer leads to the reopneing of the fingers and the movement of the primer:template junction by one base pair. -DNA Polymerase are Processive Enzymes:Processivity refers to the ability of DNA polymerases to carry out continuous DNA synthesis without dissociating from the template. It greatly increases the rate of DNA synthesis.DNA polymerase binding/finding the substrate(template:primer) in solution is aslow step that takes time. A nonprocessive enzyme will add one DNTP and release before repeating the entire procedure. The binding step is rate limiting, extremely slow, so replication would be ridic slow. But in reality, most DNA polymerase are processive. That way, they can carry out many sequential enzymatic steps while skipping the binding step to template. DNA poly also has ability to slide down the DNA template strand, which increases processivity.- Many DNA Polymerases Contain a ProofreadingExonuclease: Rarely (~10-5), an incorrect nucleotide is polymerizedinto the DNA, inhibiting further synthesis due to theincorrect structure of the mispaired base.The misshapen DNA moves from the polymeraseactive site to a second enzymatic site on the proteinthat contains a 3’ to 5’ exonuclease activity. This proofreading exonuclease removes the terminal 3’base, thus restoring the correct geometry of the primer-template junction, which can move back to thepolymerase site.( When a polymerase makes a mistake,it will create an incorrect primer:template junction thatstops the DNA polymerase at that step. Thatstructure(incorrect primer template geometry) movesto a second site on the polymerase, the exonucelase(proofreading) site.Exonuclease is 3’->5’, opposite of polymerization. It’s like hitting delete on keyboard – go backwards one step. Nuclease = enzyme that cleaves a phosphodiester bond in a nucleic acid. Exonuclease does it from the very end of the molcule, but not from inside. Endonuclease cleaves from inside the molecule. Can give them directionality (5’à3’ or opposite). This reduces mistakes to 1 in 10,000,000 times. DNA repair systems(come up later), increase accuracy by another 100 fold. ))Major limit to DNA poly accuracy: the occasional flickering of the bases into the wrong tautomeric form allows them to form pairs incorrectly. Nucleases remove incorrectly base-paired nucleotides.