DNA Replication and Synthesis

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Chapter 10

• DNA Replication and Synthesis

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Human DNA

• 3 x 109 base pairs exist in the 23 chromosomes
• Requires extreme precision to replicate only 1
  error in a 10-6 would yield 3000 errors
  
• errors can lead to evolutionary changes
• DNA synthesis is semi-conservative

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Semi-conservative Replication

• DNA strand serves as template for synthesis of
  complement strand
  
• Unwind and each template strand will open so A
  will add a T by 2 H-bonds
  
• Each strand is one old strand and one is newly
  synthesized in the double helix
  semi-conservative

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2 Other Theoretical Modes of Replication

• Conservative replication 2 newly created
  strands come together and parent strands
  reassociate conserve parent helix
  
• Dispersive replication parental strand
  dispersed into the 2 new double helix following
  replication each strand has new and old
  nucleotides
• cleavage of parent strand during synthesis

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Meselson-Stahl Experiment

• Strong evidence for semi-conservative nature
• Grow bacteria in heavy 15N for several
  generations, not an isotope
  
• All bases should have 15N incorporated and then
  switch it to 14N, collect DNA and subject to
  sedimentation equilibrium or density gradient
  centrifugation
• use a gradient of CsCl and molecule will stop
  movement when equals the density of gradient
  around it

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• After 1 round of replication, isolated DNA of a
  single band of intermediate density
  
• expect if semi-conservative mode
• After 2 rounds, see one band of intermediate size
  and a lighter band, see on subsequent rounds as
  well
  
• cant rule out dispersive but when isolate DNA
  and denature it is either 15N or 14N profiles
  only not intermediate size for ssDNA

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Different Bands in Centrifugation
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Eukaryotic Replication

• Also seen in eukaryotes
• Use 3H-thymine and autoradiography
• See grains of black where the silver grains of
  the photographic emulsion have been developed
  
• See one sister chromatid with label and can see
  where crossing over has occurred
  
• Confirms semi-conservative mode

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Origins of Replication

• Places along the chromosome that opens up to
  allow replication to take place
  
• Opening yields a replication fork that is
  bi-directional moves out in both directions to
  complete synthesis
  
• Forms a replicon length of DNA replicated after
  an opening event
  
• single origin in bacteria so entire chromosome is
  replicon
  
• eventually reach the termination region called ter

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DNA Polymerase I

• Found in E coli could conduct DNA synthesis is
  a cell-free culture
  
• Required all 4 dNTPs and a DNA template
• Synthesis was as expected for semi-conservative

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Formation of the Phosphodiester Bond

• Use a dNTP the 3rd and 2nd PO4 are removed and
  the dNMP is added to the 3 OH on the previous
  nucleotide
  
• Chain elongation occurs in the 5?3 direction
  with each step providing the next free OH

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Can DNA Pol I Work In Vivo?

• Others were skeptical of whether DNA pol I worked
  in vivo because
  
• synthesis is slower than in the cell
• works better on ssDNA than dsDNA
• appeared to be able to degrade DNA as well as
  synthesis exonuclease activity
  
• Kornberg made biologically active DNA by creating
  bacteriophage ?x174 DNA that he added to cells
  and got out infectious virus

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Mutant polA I

• 1969 a mutant with no DNA pol I activity was
  isolated that was still able to synthesize DNA
  but was unable to repair damaged DNA
  
• Conclusions from studies on this mutant
• at least one othr enzyme is responsible for
  replication
  
• DNA pol I may serve a secondary function in vivo
  responsible for fidelity but doesnt make the
  entire strand

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• None of the isolated polymerases can initiate
  synthesis without an existing DNA strand or a
  primer
  
• All synthesize in the 5?3 and have 3?5
  exonuclease activity
  
• Pol I also has 5?3 exonuclease activity and is
  in higher concentrations than the others

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Polymerases

• DNA pol III synthesizes 5?3 DNA and the 3?5
  exonuclease activity is to perform proof reading
  functions
  
• DNA pol I removes the primer and fills in the
  gap, also aids in DNA repair
  
• DNA pol II, IV and V function in various aspects
  of DNA repair caused by external forces
  
• DNA pol II is encoded by a gene that is activated
  when DNA replication is disrupted at the
  replication fork

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• Active complex is holoenzyme with 10 different
  subunits
  
• Holoenzyme and several other proteins make up the
  replisome

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Unwinding DNA Helix

• Origin is oriC 245 bp made of repeating
  sequences of 9 and 13 bases
  
• DnaA is responsible for initial unwinding binds
  to the 9-mer and leads to the binding DnaB and
  DnaC to further open and destabilize helix
  
• DnaA, B and C are a helicase and uses ATP energy
  to break H-bonds
  
• Single stranded binding protein keeps strands
  from reforming
  
• Supercoiling occurs in front of the complex which
  is relieved by DNA gyrase DNA topoisomerase
  that causes single or double stranded breaks to
  relieve the coil and then seals the backbone

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Initiation of DNA Synthesis

• Requires a primer (RNA) with a free 3-OH for pol
  III to add on nucleotides
  
• Primer is 10-12 NTPs complementary to the DNA
  template added by an RNA polymerase called
  primase which doesnt require a free 3-OH
  
• DNA pol III adds nt and then later the primer is
  removed and replaced with dNTPs (DNA pol I)

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Continuous and Discontinuous Synthesis

• DNA is anti-parallel to each other, DNA pol III
  synthesizes 5?3 meaning that it reads the DNA
  template in the 3?5
  
• stand is synthesized continuously and called the
  leading strand
  
• Opening the dsDNA makes a strand that is running
  5?3 and therefore cant be read in the
  appropriate manner
  
• this strand is synthesized in a discontinuous
  manner and is the lagging strand
  
• made is small stretches each with their own
  primer and are called Okazaki fragments

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Concurrent Synthesis

• Simultaneous synthesis occurs because the lagging
  strand forms a loop so can add nucleotides at the
  same time as the leading strand
  
• 1000-2000 nt and then run into the previous
  Okazaki fragment, form a new loop, add primer and
  continue
  
• ?-subunit (sliding clamp) forms a clamp-like
  structure around DNA duplex to keep on template
  during polymerization

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Proof-Reading and Error Correction

• Occasionally a wrong nucleotide is inserted
• All DNA pol have 3?5 exonuclease activity so
  they can detect, back up and remove the wrong
  nucleotide and then move on
  
• ? subunit of pol III is exonuclease
• This is proof-reading and increases the fidelity
  so that mistakes arent introduced to the DNA

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Coherent Model
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Replication Control

• Use conditional mutations to study what would
  otherwise be a lethal mutation
  
• protein expressed under 1 condition but not at
  another condition
  
• Temperature-sensitive mutants
• permissive temperature mutation not expressed
• restrictive temperature mutation expressed

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Eukaryotic DNA Synthesis

• Similar but more complex than prokaryotic
  synthesis
  
• More complex because DNA is linear, much more DNA
  and complexed with proteins

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Multiple Origins of Replication

• See replication bubbles in the EM and each has 2
  replication forks
  
• Need more to replicate DNA as eukaryotic
  polymerases are 25X slower than the bacterial
  versions
  
• usually takes several hours

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Information from S. cerevisiae

• Autonomously replicating sequences (ARSs) make up
  the origin
  
• Replication happens in the S phase of the cell
  cycle but it appears to happen in waves using 20
  80 of the 250-400 origins at a time
  
• During G1 phase all ARSs are bound by group of
  specific proteins forming origin recognition
  complex (ORC) synthesis doesnt start
  immediately under the control of a special
  kinase that adds a PO4
• complete the initiation complex, direct unwinding
  and DNA synthesis
  
• also prevents the formation of ORC at the ARSs
  once replicated

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Eukaryotic DNA Polymerases

• Must open the strand and remove the histone
  proteins after synthesis, the nucleosome
  reforms
  
• New histone proteins synthesized in conjunction
  with DNA synthesis to make sure have enough to
  coil up the new DNA

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• 6 eukaryotic polymerases
• pol ?, ?, and ? - nuclear DNA replication
• pol ? and ? - DNA repair
• pol ? - mitochondrial DNA synthesis
• All but ? have multiple subunits each with
  function during replication

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Processitvity

• Pol ? and ? are major forms involved in
  initiation and elongation
  
• 2 of 4 ? subunits synthesize the RNA primers on
  both the leading and lagging strand
  
• add 10 ribonucleotides another subunit comes in
  and adds 20-30 dNTP
  
• Pol ? has low processivity deals with the
  length of DNA synthesized before dissociating
  from the template
  
• Primer is in place and then undergo polymerase
  switching and pol ? comes in and elongates the
  DNA high processivity
  
• uses 3?5 for proof-reading
• Pol ? is similar to pol ? but may be restricted
  to the lagging strand
  
• Still need to remove the primer and ligate the
  backbone

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Linear Chromosomes

• Ends of linear chromosomes are problematic
  because there is no place to attach the dNTPs
  once the primer is removed on the last Okazaki
  fragment
• Ends have telomeres to help overcome this problem
  long stretches of repeats bound to specific
  telomere associated protein
  
• preserves integrity and stability of the
  chromosome

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Telomerase

• Unique enzyme that adds several repeats to the 3
  end of the lagging template strand helps
  prevent chromosomal shortening
  
• Forms a hairpin loop and can act as substrate for
  DNA pol fill in gap and cleave off the loop
  
• Telomerase is a ribonucleoprotein short RNA
  portion that is crucial to catalytic activity
  
• use RNA as a template for DNA acts as a reverse
  transcriptase

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DNA Recombination
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Gene Conversion

• Consequence of DNA recombination
• Characterized by non-reciprocal genetic exchange
  between closely linked genes in yeast and
  Neurospora
  
• Create gene mutations when fix a basepair
  mis-match