Biochemistry 441 Lecture 12 Ted Young March 6, 2000

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Arts and science are not cast into a mould, but
are formed and perfected by degrees, by often
handling and polishing, as bears leisurely lick
their cubs into form. Michel de Montaigne
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Biochemistry 441 Lecture 8Ted YoungJanuary 25,
2008

• Topic DNA replication enzymes

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Six enzymes to know-substrates, products,
co-factors, and function in DNA metabolism

• DNA polymerase
• b-clamp/processivity factor
• DNA ligase
• DNA helicase
• Single-strand binding protein
• Telomerase

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Finding genes/proteins involved in DNA replication
Biochemical approach 1. Devise an assay (what
function are you looking for?) 2. Break open
cells and apply assay 3. Demonstrate that
the protein identified plays an important role in
vivo.

• Genetic approach-
• 1. isolate temperature-sensitive (ts) mutants.
• 2. screen for DNA synthesis at the restrictive
  temperature
• 3. characterize mutants defective in DNA
  synthesis
• 4. Identify biochemical defect

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Deoxyribonucleotide polymerization
Note that Mg plays an essential Role in
catalysis.
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DNA pol I
E. coli

• Genetic evidence that pol I is not the DNA
  replicating enzyme in bacteria

mutagenesis
make crude cell-free extracts from
2000 survivors (most have a mutation in some
gene)
Measure DNA poly- merase activity in crude cell
extracts
Colony 1856 no DNA polymerase activity! But it
grows normally.
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Comparison of bacterial DNA polymerases
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E. Coli DNA pol III is a complex enzyme!
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The b-clamp-a processivity factor for polIII
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DNA replication occurs in stages .

• Initiation at a specific site called the
• origin of replication (oriC in E. coli)

oriC region
Initiation consists of several steps -binding of
dnaA protein to 9 bp repeats -binding of HU
protein to dnaA and DNA -with ATP present this
causes unwinding of the AT-rich 13 bp
repeats -DNA helicase B is loaded onto the
unwound region on both strands and begins
unwinding the helix, creating two replication
origins.
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DNA helicase
DNA helicases are directional

• DNA helicases unwind duplex DNA in the presence
  of ATP, producing single stranded DNA, AMP, and
  pyrophosphate.

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ATP
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AMP PPi
What, if anything, keeps the two complementary
strands from reforming the duplex? Essential to
unwind DNA at the origin.
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DNA single-strand binding proteins

• DNA single-strand binding proteins keep
  complementary single-strands of DNA from
  re-annealing. No energy is required.

denature
ss binding protein-
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Cooperative binding of ss DNA binding proteins
(SSBs)
Sigmoidal shape of the binding curve
indicates that binding of one molecule makes it
easier to bind the next, and so on...

• ss DNA binding proteins have a high degree of
  cooperativity binding of one protein makes it
  easier to bind a second protein.

Binding
protein
Essential function is to bind to the lagging
strand template.
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Cooperative binding (cont)

• Cooperativity requires a conformational change in
  the shape of the protein that only occurs upon
  DNA binding.

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Synthesis of Okazaki fragments
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Simultaneous continuous and discontinuous
synthesis at a replication fork by a dimeric DNA
pol III
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DNA ligase

ligase

• DNA ligase becomes activated during the reaction,
  conserving the energy of ATP or NAD as a high
  energy enzyme-adenylate intermediate.

Nicked DNA Substrate.

The reaction requires an activated- phosphorylated
-intermediate, an enzyme adenylate complex, and a
5phosphate on the DNA. RNA ligases also exist.
DNA-nick-adenylate.

ligase
Essential to link Okazaki primers to make a
continuous DNA strand
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Termination of replication in bacteria
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Termination of replication

• Circular chromosomes decatenation by
  topoisomerase(s) is required.
• Linear chromosomes a special enzyme called
  telomerase replicates the linear ends. Why is
  this necessary? Because RNA primers are at the 5
  ends. After their removal, no DNA polymerase can
  add dNTP to these ends.

The end problem in replication of linear
chromosomes
replication
5 3
Rt
RNA
Chromosomal termini, or telomeres
Left
RNA
5 ends of last Okazaki fragments
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Replication of chromosome ends by telomerase
Note that the 3 end of the chromosome is not
base-paired to DNA
(the template)
(5TTGGG-3)n N300-500

• Telomerase consists of an RNA template-telomerase
  RNA-and several proteins. The RNA acts as a
  template for the polymerase activity. The
  polymerase copies RNA into DNA, as does viral
  reverse transcriptase, extending the G-rich
  strand. The protein component of telomerase has
  homology to viral reverse transcriptases

Ch 26.3 Fig 26.35
Telomerase extends the single-stranded 3 end,
allowing Primase and DNA pol. to copy the
extended 3 end. The 3 end has redundant
sequence information so even if some is lost at
each replication, unique genetic information
isnt lost until all redundant sequence
information is lost.
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Telomeric sequences can form unique structures

• G-quartets can form both intra- and interstrand.
• Do they form in the cell??

Intra-strand G-quartet
Dimeric G-quartet
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Unique telomere structures at the ends of
chromosomes
2. Lariats can be formed from telomere-like
DNA duplexes made in the test tube in the
presence of a protein, Trf2, that is found at the
telomeres and they and are observed in the EM
after treatment of cells with psoralen (an agent
that forms inter- strand crosslinks), isolating
the DNA, and fragmenting it with restriction
endonucleases.
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Summary

• DNA replication is very complex!
• Four distinct operations must occur
• initiation of the leading strand
• initiation of the lagging strand
• elongation
• termination
• Enzyme complexes are involved at each step of the
  process
• Termination of replication by telomerase requires
  a novel type of enzyme composed of both RNA and
  protein.
• Replication is tightly coupled to the cell cycle
  and only occurs in S phase.