MCB 502A-2014. Lecture #6. Enzymatic synthesis of DNADNA polymerization directionality-1DNA polymerization directionality-2DNA polymerization directionality-3DNA polymerization directionality-4DNA polymerization directionality-5Why 5'—>3'?-1Why 5'—>3'?-2Why 5'—>3'?-3Why 5'—>3'?-4Why 5'—>3'?-5Why 5'—>3'?-6Why 5'—>3'?-7Why 5'—>3'?-8The proofreading activity of Kornberg polymerase-1The proofreading activity of Kornberg polymerase-2The proofreading activity of Kornberg polymerase-3The proofreading activity of Kornberg polymerase-4The proofreading activity of Kornberg polymerase-5The proofreading activity of Kornberg polymerase-6Slide 21The two nucleotide binding sites of the enzyme-1The two nucleotide binding sites of the enzyme-2The two nucleotide binding sites of the enzyme-3The two nucleotide binding sites of the enzyme-4The two nucleotide binding sites of the enzyme-5The DNA polymerization rate and the processivity of Kornberg enzyme-1The DNA polymerization rate and the processivity of Kornberg enzyme-2The DNA polymerization rate and the processivity of Kornberg enzyme-3The DNA polymerization rate and the processivity of Kornberg enzyme-4The DNA polymerization rate and the processivity of Kornberg enzyme-5The DNA polymerization rate and the processivity of Kornberg enzyme-6The DNA polymerization rate and the processivity of Kornberg enzyme-7PowerPoint PresentationIs Kornberg DNA polymerase a replicative enzyme?-1Is Kornberg DNA polymerase a replicative enzyme?-2Is Kornberg DNA polymerase a replicative enzyme?-3Is Kornberg DNA polymerase a replicative enzyme?-4Is Kornberg DNA polymerase a replicative enzyme?-5Autoradiography of replication forks-1Autoradiography of replication forks-2Autoradiography of replication forks-3Autoradiography of replication forks-4Another scheme of the Prescott-Kuempel experimentAutoradiography of replication forks-5Autoradiography of replication forks-6Screening for dna-minus mutants-1Screening for dna-minus mutants-2Screening for dna-minus mutants-3Screening for dna-minus mutants-4Screening for dna-minus mutants-5Screening for dna-minus mutants-6Screening for dna-minus mutants-7Screening for dna-minus mutants-8Screening for dna-minus mutants-9Screening for dna-minus mutants-10Smart GeneticsEnrichment for dna-minus mutants-1Enrichment for dna-minus mutants-2A scheme of enrichment for dna minus mutantsEnrichment for dna-minus mutants-3Enrichment for dna-minus mutants-4Enrichment for dna-minus mutants-5Enrichment for dna-minus mutants-6Enrichment for dna-minus mutants-7Enrichment for dna-minus mutants-8Cloning of genes identified by conditional mutations6. Concepts learnedHave a nice day!MCB 502A-2014. Lecture #6. Enzymatic synthesis of DNA— DNA polymerization directionality— Why 5'—>3’?— The proofreading activity of Kornberg polymerase— The two nucleotide binding sites of the enzyme— The DNA polymerization rate and the processivity of Kornberg enzyme— Is Kornberg DNA polymerase a replicative enzyme?— Autoradiography of replication forks suggests 3’—>5’ DNA synthesis and additional DNA polymerases— Screening for dna-minus mutants— Enrichment for dna-minus mutantsRan out of time and did not show the last seven slides in 2014DNA polymerization directionality-1— Is enzymatic DNA synthesis directional, and if yes, is there a preferred direction? — As you know, DNA degradation can proceed in both the 3'—>5’ and the 5'—>3’ directions along the DNA strands. — Moreover, there are enzymes that can degrade DNA strands in both directions, like ssDNA-specific ExoVII. — There are even enzymes, like ExoV, that can degrade both strands of the same DNA duplex at the same time. — If DNA polymerization is anywhere similar to DNA degradation, then a priory we can assume that there are also three possibilities for the DNA polymerization directionality: 3'—>5’, 5'—>3’; both.5' 3'5' 3'5' 3'3' 5'DNA polymerization directionality-2— To determine the directionality of DNA polymerization, one needs primed DNA templates that can be extended in one direction only: in either 5'—>3’ or in 3'—>5’. — There are at least two ways to generate such primed templates: 1) by terminal nucleotidyl transferase, which can synthesize long homopolymers, but can also synthesize combinations of different homopolymer stretches: 5' 3'5' 3'5'3'+TNTTNT3'3'3'DNA polymerization directionality-3— So, it can also be used to synthesize single long nucleotides of the following structure: 5’-dA4000dT100-3’ or 5’-dA100dT4000-3’. — There will be 3’-end primers available in the former substrate, while 5’-end primers available in the latter. 2) by a partial hydrolysis of a duplex DNA with exonucleases of known polarities. — For example, ExoIII processively degrades the 3’-ending strands of duplex DNA and, thus, generates 5’-overhangs. — Alternatively, ExoVIII processively degrades the 5’-ending strands of duplex DNA and, thus, generates 3’-overhangs.dA4000dT100dT4000dA1005' 3' 5' 3'5'3'5'3'Template-primer pairs5' 3'3' 5'5' 3'3' 5'ExoIIIExoVIIIDNA polymerization directionality-4The results of Kornberg polymerase reaction with the four templates are:Template + primer Paired end ActiondA100dT40005’ no synthesisdA4000dT1003’ synthesisExoVIII-treated dsDNA 5’ little synthesisExoIII-treated dsDNA 3’ significant synthesis— Thus, whenever there is 3’-paired end, there is DNA synthesis, but whenever there is a 5’-paired end, Kornberg polymerase cannot synthesize the complementary DNA strand. — Therefore, this enzyme can synthesize DNA in the 5'—>3’ direction only. 5' 3'5'3'DNA polymerization directionality-5— One could arrive at the same polarity indirectly, by determining the polarity of the strange exonuclease activity of Kornberg DNA polymerase (ExoVI) that is stimulated by DNA synthesis. — The nick-translation reaction propagates the nick along the DNA by employing simultaneous DNA degradation and re-synthesis going in the same direction. — Please recall that this exonuclease activity, although much inhibited, is still detectable in the absence of triphosphates. — Its polarity is 5'—>3’ on dsDNA, so the polarity of DNA synthesis must be also 5'—>3’. Time (min)% TCA-soluble 3H-countsNo dNTPs+ dNTPs14C-dTTP5' 3'5' 3'ExoVI activityWhy 5'—>3'?-1— Could we figure out the polarity of DNA synthesis by isolating precursors of DNA synthesis from cells (or determining them
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