1. Announcements
First lab report deadline extended by one week: X-linked cross lab report due 11/ 5,6.
Get lab overview for PCR lab - quiz in lab this week
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).
Group B presentations in lab 10/29, 30 - get sources approved
Monk papers will be handed back after lecture in the lab - avg. 67/75 = 89%.
Currently: reading chapters 12, 13 this week.

2. 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

3. Outline of Lecture 22 I. Eukaryotic DNA replication is more complex
II. The “end” problem and telomerase
III. DNA Recombination
IV. The Genetic Code

4. I. Multiple Replication Forks During Eukaryotic DNA Synthesis

5. When, during the cell cycle, can new replication origins be formed?
G 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

6. 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

7. 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???

8. 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.

9. 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.

10. DNA Recombination (12.20a-f)
A B
Heteroduplex DNA
Branch
migration
a b
Can occur all the way to
the end or second pair of
nicks can create internal
recombinant fragment.
Nicking
Displacement
Holliday structure
Ligation

11. DNA Recombination (12-20f-g)
EM Evidence for
Mechanism

12. DNA Recombination (12.20h-i)
Recombinant duplexes formed b a
Nicks here would create noncrossover
duplexes
Exonuclease nicking

13. 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)?

14. 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.

15. 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.

16. Frameshift Mutations Garble the Code, Leading to Mutant Protein
Produces normal protein
Produces mutant protein
May or may not produce
a normal protein.

17. 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

18. 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

19. 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-...

20. 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-...

21. 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.

22. 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.

23. Note that 3rd Base Position is Variable

24. Degeneracy and the Wobble Hypothesis
Codon in mRNA
Anticodon in tRNA
Codon: 5’ ’
Anticodon: 3’ ’
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”)

25. Special Anticodon-Codon Base-Pairing Rules