Week 9: DNA Polymerase + Replication

fractionation

activity assay to find which fraction has DNA polymerase in it

1. grow E.coli in large amounts w/ protein of interest

2. break open cells

3. prepare soluble extract

4. fractionate/separate extract to resolve different proteins → repeat

   1. can do based on size, solubility, etc.

5. confirm activity via activity assay

   1. look for incorporation of radioactivity in polymerized DNA

      1. polymerized DNA has different solubility than free NTs

Kornberg experiment

experiments that demonstrated enzymatic synthesis of DNA + identified DNA polymerase as enzyme responsible for DNA replication → DNA Pol I

1. mixed radio-labeled dT or dTTP w/ isolated fraction

2. added acid to reaction + separated into 2 fraction via centrifugation:

   1. acid soluble

   2. acid insoluble

3. acid insoluble = could be polymerized DNA → polymerase is present in mixture

4. verified acid insoluble pellet contained polymerized DNA by treat w/ Dnase

   1. Dnase specific to DNA → cleaves phosphodiester backbone → all NTs free

      1. real nucleotide polymers broken up → soluble again in acid conditions

      2. radioactive moves from pellet into soluble fraction

   2. NTs could be stuck in middle of protein aggregations/interact w/ protein

      1. need to discern b/w free stuck NTs or free NTs from DNA

DeLucia + Cairns

• isolated mutant E.coli w/ no DNA pol I activity

  • DNA pol I = prototype for all DNA polymerase

    • enzymatically + structurally

Nicholas Kornberg

discovered DNA pol I through series of fractionation experiments

Tom Kornberg

discovered DNA pol III

• polymerase responsible for replicating E.coli chromosome in vivo

DNA polymerase I

involved in cleanup during replication/recombination/repair

• abundant but insufficient for replication of E.coli chromosome

  • rate = 600 NT/min

    • slower than observed for replication fork movement

  • low processivity → falls off easily

• 3’→5’ exonuclease activity = proofreading

• 5’→3’ exonuclease = nick translation

  • removes RNA/DNA hybrids made by primase

    • remove RNA primer + add DNA behind

DNA polymerase II

involved in DNA repair

• slow polymerization rate

• intermediate processivity

• 3’→5’ exonuclease activity = proofreading

DNA polymerase III

principal replication enzyme in E.coli

• 3’→5’ exonuclease activity = proofreading

• fast polymerization rate

• high processivity

DNA polymerase IV + V

involved in specialized DNA repair

• no exonuclease activity in 3’→5’ or 5’→3’ direction

• very low polymerization rate = 1-3 NT/s

• very low processivity

nick translation

break/nick in DNA phosphodiester backbone moved along w/ enzyme

• 5’ → 3’ exonuclease activity

  • carried out by larger or Klenow fragment in DNA pol I

    • makes nicks + removes nucleotides as enzyme moves in 5’→3’ direction

    • normal polymerase active site adds NTs onto 3’OH end of strand

• important in

  • DNA repair

  • removal of RNA primers during replication

3 to 5 exonuclease

proofreading function

• polymerase reads “backwards”

• DNA synthesized 5’ → 3’ direction

  • wrong base added

  • read backwards along sequence to remove wrong base

5 to 3 exonuclease

nick translation function

• aka large fragment or Klenow fragment

  • domain in front of enzyme + performs nick translation

• only in DNA pol I

• mild protease treatment separates domain from enzyme

DNA polymerase

synthesizes new DNA polymers in template driven process in 5’→3’ direction

requires:

1. single-stranded template

2. deoxyribonucleotides w/ 5’ triphosphate = dNTPs

3. Mg2+ ions = co-factor for polymerase

   1. aspartic acid residues = (-)

   2. Mg2+ has to be in right spot to stabilize transition state at 3’OH

   3. catalyzes reaction

4. annealed primer w/ free 3’OH

   1. often RNA

DNA polymerase reaction mechanism

0. primer already bound to DNA template strand

1. incoming dNTPs form WC bonds w/ template strand

2. coordination w/ Mg2+ stabilizes 3’ OH + a-phosphate

3. new bond forms b/w a-phosphate of incoming NT + 3’OH of primer

4. beta + gamma phosphate of NT released as PPi new phosphodiester bond forms

5. as long as template strand is available → new substrate generated for next round of polymerization

6. once last NT reached → stops

DNA polymerase template

guides polymerization according to WC base-pairing rules

• needs to be single-stranded

  • double-strand → all H-bonds satisfied = can’t add more NTs in

    • need helicase to unwind

DNA helicase

unwinds parental DNA to make single-stranded template

in vitro assay

• radio-label 1 strand of dsDNA fragment

• add purified enzyme in gradient = ⬆ [enzyme]

• look for ability of protein to unwind DNA via ssDNA product formation

  • DNA segments become smaller as ⬆ [enzyme]

single-stranded binding protein (SSBP)

binds to single-stranded DNA to prevent spontaneous re-annealing + intra-strand interactions

• in E.coli = SSP

• in humans = RPA

• sequence-independent = doesn’t make specific contact w/ DNA

• needs to be jiggly = allow polymerase to read DNA

shape discrimination

contributes to fidelity of DNA replication via base-pair geometry

• active site flexible enough for catalysis of properly matched bases

• incorrect BP = binding site contorts = less favourable

  • prevents replication of wrong BPs

DNA polymerase error correction (proofreading)

translocation of enzyme inhibited when wrong NT added

• 3’→5’ exonuclease activity → removes newly added NT

  • domain flips to active site

  • mismatch fits into space for mismatched bases

    • favours non-Cannonical BPs

  • exonuclease cleaves off NT

semi-conservative replication

parent + daughter strands wind together

• daughter strands different from e/o but net product w/ parent = identical copies

Meselson-Stahl experiment

1. grow E.coli in “heavy” nitrogen environment to label DNA

   1. bacteria copy entire DNA complement (genome) before every cell division

   2. DNA = heavy

2. moved into normal N-containing medium

   1. cell divide once more

   2. DNA = lighter

3. separate DNA by density via cesium chloride density gradient

   1. heavy → travels more

4. results support semiconservative replication

   1. after 1 generation = DNA intermediate

      1. conservative replication ❌

   2. after 2 generation = DNA either all light or intermediate

      1. semiconservative replication ✅

replication fork

dynamic points where parent DNA is unwound + separated strands are replicated

• both strands replicated simultaneously

• both ends of bacterial chromosome have active replication forks → bidirectional replication

denaturation mapping

• selective denaturing of sequences rich in A-T base pairs

  • AT = only 2 H-bonds + ⬇ base stacking = easier to melt (separate)

  • provide landmarks along DNA molecule

    • generates reproducible pattern of single-stranded bubbles

origin

location where replication loops are initiated

bidirectional replication

process where DNA replication occurs simultaneously in both directions from single origin of replication, resulting in two active replication forks

semi-discontinuous replication

mode of DNA replication where 1 strand (leading strand) is synthesized continuously while other strand (lagging strand) is synthesized in short segments (Okazaki fragments) due to the antiparallel nature of the DNA double helix

Okazaki’s experiment

method

1. pulsed E.coli w/ radioactive thymidine

   1. newly synthesized DNA labeled = “hot'“

2. used alkaline sucrose density gradient

   1. separate DNA based on size

3. assay fractions from centrifugation experiment at various time points

   1. DNA synthesis stops once cells are lyzed

   2. can see “snap” shots of reaction

results

• trace = time point

• dot = point along gradient density

• further down gradient = larger fragment w/ “hot” Thymidine

• over time → see longer DNA fragments + still see short ones

• supports discontinuous replication model

DNA polymerase III clamp loader

loads clamp onto DNA-RNA hybrid

• requires ATP binding + hydrolysis

  • ATP physically opens up ring

  • ring snaps back around region

• helps w/ processivity of polymerase

leading + lagging strand synthesis

1. helicase (DnaB) unwinds DNA at replication fork

   1. single-stranded lagging strand coated by SSBP

2. leading strand synthesized in one piece

3. lagging strand synthesized in fragments = Okazaki fragments

4. primase = adds primer to make free 3’OH for polymerase

5. clamp loader loads on clamp

   1. ensures synthesis is processive

6. lagging strand synthesized until primer on older Okazaki fragment reached

7. clamp loader reloads clamps on new primer/template RNA-DNA hybrid until synthesis completes

8. DNA pol I removes RNA primer + replaces w/ DNA via nick translation (5’→3’ exonuclease)

9. DNA ligase seals remaining nick in phosphodiester backbone