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									                     Announcements
1. First lab report deadline extended by one week: X-linked cross lab
   report due 11/ 5,6.

2. Get lab overview for PCR lab - quiz in lab this week

3. If your transformation did not work, look at plates in lab refrigerator,
   both groups “A” and “B”; make counts from these plates. Lab
   assignment due 10/29, 30 (extra week).

4. Group B presentations in lab 10/29, 30 - get sources approved

5. Monk papers will be handed back after lecture in the lab - avg.
   67/75 = 89%.

6. Currently: reading chapters 12, 13 this week.
         Review of Last Lecture

I. Bacterial DNA replication - semiconservative

II. Many complex issues to resolve during DNA replication
       - DNA helix must be unwound, RNA primer needed
       - Synthesis is both continuous and discontinuous
       - Proofreading is critical
          Outline of Lecture 22
I. Eukaryotic DNA replication is more complex

II. The “end” problem and telomerase

III. DNA Recombination

IV. The Genetic Code
I. Multiple Replication Forks During
     Eukaryotic DNA Synthesis
 When, during the cell cycle, can new
 replication origins be formed?

      G1                S                  G2
Pre-replication    replication        post replication

ARSs complex       DNA synthesis      DNA synthesis
Into ORCs                             completed

Pre-RCs can form   No new pre-RCs     No new pre-RCs

    ARS = autonomously replicating sequence = origin
    ORC = origin recognition complex
    Pre-RC = pre-replication complex
        Eukaryotic DNA Polymerases
Enzyme                Location         Function
• Pol  (alpha)       Nucleus          DNA replication
   – includes RNA primase activity, starts DNA strand

• Pol  (gamma)        Nucleus        DNA replication
   – replaces Pol  to extend DNA strand, proofreads

• Pol  (epsilon)        Nucleus        DNA replication
   – similar to Pol , shown to be required by yeast mutants

• Pol  (beta)         Nucleus         DNA repair
• Pol  (zeta)         Nucleus         DNA repair
• Pol  (gamma)        Mitochondria    DNA replication
II. The Eukaryotic Problem of Telomere Replication


                                   RNA primer near
                                   end of the
                                   chromosome on
                                   lagging strand
                                   can’t be replaced
                                   with DNA since
                                   DNA polymerase
                                   must add to a
                                   primer sequence.

                               Do chromosomes get
                               shorter with each
                               replication???
Solution to Problem: Telomerase

                • Telomerase enzyme adds
                  TTGGGG repeats to end of lagging
                  strand template.



                • Forms hairpin turn primer with free
                  3’-OH end on lagging strand that
                  polymerase can extend from; it is
                  later removed.

                • Age-dependent decline in telomere
                  length in somatic cells, not in stem
                  cells, cancer cells.
III. Recombination at the Molecular Level

 • Breakage and joining also directed by enzymes.

 • Homologous recombination occurs during synapsis in
   meiosis I, general recombination in bacteria, and viral
   genetic exchange.

 • Molecular mechanism proposed by Holliday and
   Whitehouse (1964).

 • Depends on complementary base pairing.
    DNA Recombination (12.20a-f)
A       B
                              Heteroduplex DNA

a       b          Branch
     Nicking       migration Can occur all the way to
                              the end or second pair of
                              nicks can create internal
                              recombinant fragment.

    Displacement

                            Holliday structure

     Ligation
 DNA Recombination (12-20f-g)

EM Evidence for
    Mechanism
        DNA Recombination (12.20h-i)
A
            B
                 Recombinant duplexes formed

    b
                 a



                Nicks here would create noncrossover
                duplexes


          Exonuclease nicking
IV. Early Evidence for the Genetic Code

 • 1940’s: Beadle and Tatum noted correlation between gene
   mutation and nonfunctional enzyme

 • First direct evidence: sickle-cell hemoglobin
    – single nucleotide change > change in amino acid


 • 1961: Jacob and Monod proposed that mRNA is an
   unstable intermediate between DNA and protein

 • How could four letters (A, T, G, C) spell out 20 words (the
   amino acids)?
           Theoretical Evidence

• Sidney Brenner (early 1960’s) argued
  that code must be triplet theoretically.

• If a two letter code, how many amino
  acid “words” could be made from A, U,
  G, C? 42 = 16

• If a three letter code, how many “words”
  could be made? 43 = 64, more than
  enough for the 20 amino acids.
Genetic Evidence: Frameshift Mutations

• 1961: Francis Crick, Barnett, Brenner, and Watts-Tobin

• Created insertion and deletion mutants in cistron B of rII
  locus of phage T4
   – A cistron codes for a single polypeptide chain within a gene


• Proflavin (a DNA dye) was used as a mutagen.

• Proflavin caused insertion or deletion of one or more
  nucleotides in the cistron, usually causing a frameshift of
  the putative genetic code.
Frameshift Mutations Garble the Code,
     Leading to Mutant Protein

                       Produces normal protein



                       Produces mutant protein




                        May or may not produce
                        a normal protein.
        Wildtype, Single Insertion and
                   Deletion
5’ UGC GAA AAC ACA AGA GCA UUA U 3’        WT
    C   E   N   T   R   A      L
                    Functional Site
               
5’ UGC GAA AAC GAC AAG AGC AUU AU 3’   +   MUT
    C   E   N   N   K   S      I

              A
5’ UGC GAA AACCAA GAG CAU UAU 3’      -   MUT
    C   E   N   Q   Q   H   Y
  Insertion/Deletion, Triple Deletion, Triple
                   Insertion
               A
5’ UGC GAA AAC GCA AGA GCA UUA U 3’    +/- WT
    C   E   N   A   R   A   L

       GAA
5’ UGC  AAC ACA AGA GCA UUA U 3’     -/-/- WT
    C       N   T   R   A   L

           
5’ UGC GAA GAA AAC ACA AGA GCA UUA U 3’ +/+/+ WT
    C   E   E   N   T   R   A   L
            Biochemical Evidence

• 1961: Nirenberg, Matthaei used synthetic mRNAs
  and an in vitro translation system to decipher the
  code.

• Polynucleotide Phosphorylase enzyme links NTPs to
  make RNA without a template

• Homopolymers:
   – poly(U) codes for Phe-Phe-Phe-Phe-…
   – poly(A) codes for Lys-Lys-Lys-Lys-…
   – poly(C) codes for Pro-Pro-Pro-Pro-...
           Repeating Copolymers

• Khorana, early 1960’s

• UGUGUGUGUGUGUGUGU...
   – Cys-Val-Cys-Val-Cys-Val-...
   – Therefore GUG or UGU codes for either Cys or Val


• UUCUUCUUCUUCUUC…
   – Phe-Phe-Phe-Phe-... or
   – Ser-Ser-Ser-Ser-… or
   – Leu-Leu-Leu-Leu-...
     In Vitro Triplet Binding Assay
• Nirenberg and Leder (1964) mixed all 20
  amino acids with ribosomes, different RNA
  triplets:
  – Ribosomes + UAU -> Tyr binds
  – Ribosomes + AUA -> Ile binds
  – Ribosomes + UUU -> Phe binds, etc.
Nucleic Acid to Protein


              • How does the information in
                codons of mRNA get
                translated into amino acids
                in polypeptides?

              • Through adapter molecules:
                tRNA

              • tRNA has anticodon that
                base pairs with the codon in
                mRNA and carries an amino
                acid corresponding to that
                codon.
Note that 3rd Base Position is Variable
Degeneracy and the Wobble Hypothesis

                 • Codon in mRNA
                 • Anticodon in tRNA
                 • Codon: 5’-1-2-3-3’
                 • Anticodon: 3’-3-2-1-5’
                 • First two bases of codon are
                   more critical than 3rd base
                 • Base-pairing rules are relaxed
                   between 3rd base of codon and
                   1st base of anticodon (third
                   base “wobble”)
Special Anticodon-Codon Base-Pairing Rules

								
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