MCB 502A-2015. 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 mutantsDNA polymerization directionality-1 — Is enzymatic DNA synthesis directional…. ? — … DNA degradation … both the 3'—>5’ and the 5'—>3’… — … ExoVII degrades in both …. — … ExoV degrades both strands … at the same time. — If DNA polymerization is similar to DNA degradation, … then … 3'—>5’, 5'—>3’; both.5' 3'5' 3'5' 3'3' 5'DNA polymerization directionality-2 — To determine the directionality… primed template, … 5'—>3’ or in 3'—>5’. — … two ways… : 1) by terminal nucleotidyl transferase, … : 5' 3'5' 3'5'3'+ TNT TNT 3'3'3'DNA polymerization directionality-3 — For example, …. : 5’-dA4000dT100-3’ or 5’-dA100dT4000-3’. — … 3’-end primers … , … 5’-end primers. 2) … partial hydrolysis of dsDNA … — … ExoIII … degrades the 3’-ending strands … — … leaving 5’-overhangs. — … ExoVIII … degrades the 5’-ending strands … — … leaving 3’-overhangs.dA4000 dT100 dT4000 dA100 5' 3' 5' 3'5'3'5'3'Template-primer pairs5' 3'3' 5'5' 3'3' 5'ExoIIIExoVIIIDNA polymerization directionality-4 The 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— … 3’-paired end, — DNA synthesis, … — Therefore, this enzyme can synthesize DNA in the 5'—>3’ direction only. 5' 3'5'3'DNA polymerization directionality-5 — …same conclusion … indirectly, … via … ExoVI… — The nick-translation reaction… — … this exonuclease activity… needs no triphosphates. — … 5'—>3’ on dsDNA, so the polarity of DNA synthesis must be also 5'—>3’. Time (min)% TCA-soluble 3H-countsNo dNTPs5' 3'5' 3'ExoVI activityWhy 5'—>3'?-1 — … figure out the polarity …by the structure of precursors…? — … precursors … have … one side "activated" … — … the monomer units within DNA are dNMPs …, while DNA precursors are dNTPs .., … which side of the dN is charged with PPP, — the 3’ or the 5’? OH O PPP-O B 5' 3' O-PPP O HO 5' 3' BWhy 5'—>3'?-2 — … in vivo, only 5’-dNTP. — … in vitro, only 5’-dNTP … — Does the invariable 5'-position of the triphosphates on the DNA precursors commit DNA synthesis to the 5’—>3’ direction? — … various polymer biosynthesis :…Head growthTail growthWhy 5'—>3'?-3 — … tail growth … — an attack of the non-activated end of a polymer on the activated end of a precursor. — Examples :… DNA, RNA and some polysaccharides. — Head growth — attack of the non-activated end of a precursor on the activated end of a polymer. — Examples :… protein synthesis, fatty acid biosynthesis and synthesis of O-antigen.Why 5'—>3'?-4 — … no big reason why DNA should be growing tail-first (5’—>3’) rather than head-first (3’—>5’). — … one nuance …to prefer the 5’—>3’ DNA polymerization … if dNTPs are 5’-PPP. — … irreversible polymerization … pyrophosphate hydrolysis …. — The 3’—>5’ chain growth is still an option. 5' 3' 5' 3'Why 5'—>3'?-5 — … once in a while, … DNA polymerase inserts a wrong nucleotide…. — … all DNA polymerases have … proofreading exonucleases… . — … backs down … and hydrolyzes the last incorporated nucleotide. — (Pyrophosphorolysis is not an option, …). — … tail-first, — … ready to go. 5' 3' 5' 3'Why 5'—>3'?-6 — … head-first, … proofreading … the 5’-end is not ready … — … needs to be charged with a pyrophosphate… — … with all DNA precursors 5’-dNTPs and the concentration of pyrophosphate low to make DNA replication irreversible, … 5' 3' 5' 3'Why 5'—>3'?-7 — … transcription … has no proofreading, … — The "transcription" argument against is weak… as the original replicative RNA-dependent RNA polymerases… Negative stranded RNA virus replication http://viralzone.expasy.org/all_by_species/1096.htmlWhy 5'—>3'?-8 — Sadly, … impossible to test, — … another example of "non-science".The proofreading activity of Kornberg polymerase-1— In addition to … Kornberg polymerase has the 3’—>5’ exo … against both duplex and ssDNA. — … with the … substrate (poly-dA:oligo-dT) in the absence of dNTPs, … on dsDNA this exonuclease activity is completely inhibited by DNA precursors… .— … opposite to DNA polymerization, — … a mechanistic reversal of polymerization? 5'5'5'5'5'5'K-pol K-pol +dNTPThe proofreading activity of Kornberg polymerase-2— … no, because pyrophosphate is not required…— Besides, pyrophosphorolysis is inactive against ssDNA, …— … released nucleotides … are dNMPs…OH O PPP-O B 5' 3' OH O P-O B 5' 3'The proofreading activity of Kornberg polymerase-3— The substrate to distinguish between 3’—>5’hydrolysis and pyrophosphorolysis …: dA4000:dT300-dC(3H)… — Pyrophosphorolysis is completely inhibited …— In contrast, the 3’—>5’ exo… is faster…dA4000dT300-dC(3H)5'3'dA4000dT300-dT(3H)5'3'% TCA-precipitable 3H-countsTime (min)100%The proofreading activity of Kornberg polymerase-4— … dNTP challenge…— … poly-dA:oligo-dT-(14C-dT)5-(3H-dC)16 with various dNTPs… to stop the degradation. — The degradation of the 3H-dC-tail in cannot be prevented by any dNTP, … dA4000dT300-dT5(14C)-dC15(3H)5'3'% TCA-precipitable 3H-countsTime (min)100%14C, +dTTP3H, +dTTP14C, no dTTP3H, no dTTPThe proofreading activity of Kornberg polymerase-5— … the biological significance …? — It cannot be just degradation, …— there are always dNTPs in the cell. — … can Kornberg enzyme extend a template with a mispaired 3’-end…? — … it can, … after all the 3H radioactivity… dA4000dT300-dC16(3H)5'3'% TCA-precipitable 3H-countsTime (min)100%dTTP(14C)14C3HThe proofreading
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