Discovery of DNAP - I.R. Lehman

discovery

• Kornberg, Lehman, Bessman, Simms

• discovery of the first DNA polymerase, DNA polymerase I of Escherichia coli from in-vitro experiment

• simple and quantitative assay in E.coli extract → enzyme purification to elucidate the reaction(s) catalyzed

• Initially declined by J of Biological Chemistry → later published in Proceedings of National Academy of Science

What is the significance of the [14C]thymidine being converted to an acid insoluble product?

• incorporation of labeled thymidine into an acid-insoluble product provided evidence for formation of DNA synthesis in vitro: thymidine incorporated into a macromolecular structure resistant to solubilization in trichloroacetic acid (TCA) → macromolecular structure likely DNA.

  • [14C]thymidine converted to an acid-insoluble product by an extract of log-phase E. coli in the presence of ATP

  • The fact that the radioactivity becomes acid-insoluble suggests that the labeled thymidine has been incorporated into a stable, insoluble structure, which is a characteristic feature of DNA

• acid-solubility test using crystalline pancreatic DNase further supports the idea that the product is associated with DNA → treatment with DNase renders all of the radioactivity acid-soluble → indicates that labeled thymidine has been integrated into a structure susceptible to DNase digestion

Why does DNase convert the product to an acid-soluble form?

To confirm that the acid-insoluble product is likely a larger structure, possibly DNA, and the treatment with DNase breaks it down into smaller fragments that are soluble in acid

• allowed to confirm the incorporation of labeled thymidine into DNA by making the radioactivity acid-soluble after DNase treatment.

Why was the thymidine labeled with 14C?

• To track its incorporation into DNA

• exclusive to DNA

Is thymidine a direct precursor for DNA? What is?

Yes

Why was [32P]dTMP better than [14C]thymidine?

• increased specific radioactivity → allowed to detect lower concentrations of the labeled dTMP

• improved radioactive count → easier to quantify incorporation of labeled thymidine into the acid-insoluble product

What was the true substrate for the enzyme found (thymidine kinase)

dTTP (rather than dTMP or dTDP)

what was the ratio of thymidine:deoxyribose:phosphate in dTTP?

1:1:3

What did their final assay mixture consist of?

• crude sonic extract of E. coli

• 𝜶-[32P]dTTP

• ATP

• Mg2+

• buffer

What was streptomycin sulfate used for?

• to separate nucleic acids from nucleotides → precipitate w/ cellular nucleic acids (S-fraction) + nucleic acid-free supernatant (P fraction)

  • +ve charge allowed to precipitate -vely charged nucleic acids

  • nucleic acids → hindrance to protein purification in bacterial extracts

What can you infer from the observation that neither fraction ‘S’ nor fraction ‘P’ could support DNA synthesis on their own, whereas the two fractions combined could?

Each component plays a specific role, and absence of any one component diminishes activity → need all factors for efficient DNA synthesis

• Multiple Enzymes Required from both fractions:

  • DNA polymerase (catalyzed phosphodiester bond synthesis)

  • deoxynucleotide kinases

  • nucleoside-diphosphate kinase

• requirement for two heat-labile fractions (S+P), a heat-stable fraction (P-fraction), three heat-stable dialyzable chromatographically distinct fractions (s-fraction), and ATP for successful incorporation of dTTP

  • heat-labile component (P fraction): DNA polymerase

  • heat-stable, non-dialyzable component (P fraction): DNA

  • heat-labile, non-dialyzable component (S fraction): mixture of deoxynucleotide kinase + nucleoside-diphosphate kinase → heat-stable, dialyzable mixture of dCTP, dATP, and dGTP

P-fraction

• showed no dTTP incorporation ino DNA (no activity)

• But, prior incubation of the extract for a few minutes at 37 °C increased activity

What was suggested by heat-labile fractions (as opposed to heat-stable ones)?

they were enzymes who’s activity depended on temperature

• P fraction: DNA polymerase

• S fraction: kinases

What were the different functions served by the DNA in the extract?

• source of dNMP (deoxynucleoside monophosphate): precursors for dNTP

• protected the labeled DNA synthesized from degradation by the nucleases in the extract → ensured integrity and stability of newly synthesized DNA → accurate analysis of the incorporation of labeled dTTP

• primer for DNA chain extension through addition of dTMP from dTTP to pre-existing DNA chains (existing DNA chains provided a template for synthesis of new DNA strands)

How were the four 32P-dNTPs prepared?

32P-labeled DNA, isolated from 32 P-labeled E. coli → treatment with pancreatic DNase and snake venom phosphodiesterase → 32P-dNMPs (dAMP, dCMP, dTMP, and dGMP) → 32P-dNTPs

What is an ion exchanger, and how is it used in fractionation?

• chromatography technique for separating & purifying ions based on their charge properties → used to isolate DNA polymerase

  • contain charged functional groups → selectively interact with ions of opposite charge → separation of different ions

  • e.g., DEAE-cellulose and phosphocellulose

Why couldn’t they remove all nuclease activity from their purified polymerase?

presence of 3’→5’ exonuclease activity functionally associated with all DNA polymerase (proofread and excise incorrectly incorporated nucleotides) → removing this would interfere with normal enzymatic functions crucial for maintaining the accuracy of DNA replication

Why were all four dNTPs required for DNA synthesis? Why was this amazing?

• served as a primers for template-directed replication by DNA polymerase & elongation of DNA chain

• amazing because:

  • it supported Watson and Crick's proposed mechanism

  • ratio in the product corresponded closely to that of the added dNTPs throughout the synthesis and was independent of the relative concentrations of the individual dNTPs (didn’t affect base ratios of product)

What is the role of DNA polymerase I in replicating the E coli chromosome?

ribonuclease H + DNA polymerase activity → process Okazaki fragments during discontinuous replication of the lagging strand at the replication fork for ligation

What is the role of DNA polymerase III in replicating the E coli chromosome?

holoenzyme that catalyzes synthesis of chromosome

Why do organisms have more than one DNA polymerase?

• allows organisms to perform a range of tasks related to DNA replication, repair, and maintenance

• Each polymerase evolved to specialize in specific functions, contributing to the overall efficiency, accuracy, and adaptability of DNA metabolism in different cellular contexts

Requirements of DNA polymerase

• template (DNA or RNA)

• primer

• four dNTPs

• Mg²+

eukaryotic DNA polymerase

• DNA polymerases α, δ, and ε: nuclear DNA replication

• DNA polymerase γ: mitochondrial genome replication