I' Multiple Replication Forks During Eukaryotic DNA Synthesis

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I. Multiple Replication Forks During Eukaryotic
DNA Synthesis
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When, during the cell cycle, can new replication
origins be formed?
G1 S G2 Pre-replication replica
tion post replication ARSs complex DNA
synthesis DNA synthesis Into ORCs completed P
re-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
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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

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II. The Eukaryotic Problem of Telomere Replication
RNA primer near end of the chromosome on lagging
strand cant be replaced with DNA since DNA
polymerase must add to a primer sequence.
Do chromosomes get shorter with each
replication???
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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.

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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.

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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
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DNA Recombination (12-20f-g)
EM Evidence for Mechanism
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DNA Recombination (12.20h-i)
A
B
Recombinant duplexes formed
b
a
Nicks here would create noncrossover duplexes
Exonuclease nicking
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IV. Early Evidence for the Genetic Code

• 1940s Beadle and Tatum noted correlation
  between gene mutation and nonfunctional enzyme
• First direct evidence sickle-cell hemoglobin
• single nucleotide change gt 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)?

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Theoretical Evidence

• Sidney Brenner (early 1960s) 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.

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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.

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

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Repeating Copolymers

• Khorana, early 1960s
• 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-...

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In Vitro Triplet Binding Assay

• Nirenberg and Leder (1964) mixed all 20 amino
  acids with ribosomes, different RNA triplets
• Ribosomes UAU -gt Tyr binds
• Ribosomes AUA -gt Ile binds
• Ribosomes UUU -gt Phe binds, etc.

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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.

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Note that 3rd Base Position is Variable
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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)

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Special Anticodon-Codon Base-Pairing Rules