DNA Replication

Question 1

Lehman DNA Polymerization Assay

Answer 1

• Used E.coli as activity source
• Added ATP, Magnesium, and C14 radiolabeled deoxythymidine to detect presence of DNA polymerization activity
• Free floating dT nucleotides would be soluble and found in supernatant
• dT nucleotides within DNA polymers would be insoluble and found in pellet after centrifugation
• Found 0.01% radioactivity in pellet
  
  • radiolabeled dT unintentionally trapped in pellet (E.coli not source of polymerization activity) or polymerization inefficient but is source of activity

Question 2

Treatment with DNAse

Answer 2

• Cuts DNA polymers into individual nucleotides
• Nucleotides moved from pellet to supernatant
• E.coli is source of polymerization activity

Question 3

Improving assay from Kornberg and Sims

Answer 3

• dTTP is more efficient substrate and P32 is more sensitive label
• phosphorylated nucleotide generated by phosphorylating dT to dTMP via thymidine kinase and ATP, dTMP to dTDP via nucleotide kinase and ATP, and dTDP to dTTP via nucleotide diphosphatase kinase and ATP
• More efficient because did not require phosphorylation before utilization in DNA polymerase
• a-phosphate of dTTP must carry radiolabel
• P32 radiolabeling done by transferring radioactive-y-phosphate group

Question 4

Purification of DNA Polymerase 1

Answer 4

• Fractionation columns separate components based on size
• Largest protein elutes fastest, second fraction is medium, smallest elutes the slowest
• Components of E.coli extract separated based of solubility using streptomycin (positively charged)
• Add radiolabeled dTTP, Mg2+, and ATP in each fraction, acid precipitation, and check pellet for radioactivity
• Process yielded two fractions (S and P)
• Neither S or P alone showed radiolabeled activity

Question 5

Control group of fractionation and findings

Answer 5

• Recombined S and P fractions to recreate the original E.coli extract
• Control did have polymerization activity
• Findings implied unique polymerization substrates in both S and P fractions -> need all substrates for polymerization activity

Question 6

Substrates

Answer 6

• DNA template found in P fraction
• all four dNTPs in S fraction
• kinases in S fraction

Question 7

Case Study

Answer 7

• One of the drugs used to treat HIV is AZT
• AZT is thymidine analogue with azide group (N3) at 3’ position of the ribose
• Prevents replication of HIV virus by preventing next base from being added onto growing DNA chain
• Does not affect eukaryotic replication because exonuclease of DNA Pol can remove altered base

Question 8

polA

Answer 8

• DNA Polymerase 1 not fast enough for genome replication
• Paula DeLucia had to individually search thousands of E.coli cells using Lehman assay
• Found DNA Polymerase 1 mutant (polA) -> showed DNA polymerase 1 is not essential for life and is not responsible for genome replication

Question 9

Discovery of DNA Polymerase 2 and 3

Answer 9

• fractionated polA mutant extract and evaluated each fraction for polymerization activity
• Led to discovery of DNA polymerase 2 and 3

Question 10

DNA polymerization

Answer 10

• 3’OH of DNA/RNA primer attacks a-phosphate of incoming dNTP to form a phosphodiester bond
• With each nucleotide addition, molecule of pyrophosphate released -> cleaved into two inorganic phosphates by pyrophosphatase
• Polymerization reaction driven forward by cleavage of high energy phosphoanhydride between phosphate groups
• Need high level of pyrophosphate to go in reverse direction

Question 11

Fidelity of DNA Polymerase

Answer 11

• Fidelity inversely related to error rate (high fidelity means low error rate)
• Error rate of DNA polymerization is 10^-10 -> one error made every 10 billion bases
• Use four key mechanisms:
  
  • Base pairing: Fidelity is 10^-2
  • Shape discrimination: incorrect bases have hard time fitting into active site of DNA polymerase. Incorrect bases that do fit are inefficiently incorporated. Decreases error rate by 10^-3
  • Proofreading: 3’-5’ exonuclease activity of DNA polymerase removes incorrectly incorporated bases, decreases error rate by 10^-2. RNA polymerase does not have proofreading activity
  • Mismatch repair: Mismatch repair pathway removes incorrect bases after DNA polymerization is complete. Decreases error rate by 10^-3

Question 12

Proofreading reaction

Answer 12

• Polymerization site (normally faster rate of rxn): (dNMP)n + dNTP <=> (dNMP)n+1 + PPi
• Exonuclease site (if incorrect base, faster rate of rxn): (dNMP)n <=> (dNMP)n-1 + dNMP
• Reactions not simply reverse of each other, require different substrates