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Today’s Outline
• Study Notes 6b Due!
• Take up exam
– View exam on Friday between 2-4pm in C-4.
– Written appeals-process
• DNA Replication
– Replication
– PCR
– Telomeres
• DNA Replication Tutorial 3-5pm Friday
Write Down:
What is one thing you learned about DNA
replication from the readings?
To Understand DNA Replication,
Understand the Parts
-
Helicase
Topoisomerase
Single Strand Binding Proteins
DNA Polymerase III
Sliding Clamp
DNA Primase
Helicase
Helicase unzips DNA.
Topoisomerase
Relieves the tension produced when DNA is
unzipped.
Single Strand Binding Proteins
Stabilizes DNA and prevents it from binding to
parallel strand.
DNA Polymerase III
Adds dNTPs to the newly synthesized strand on
the leading strand. Adds in a 5’  3’ direction.
Sliding Clamp
Holds DNA Polymerase III in place.
DNA Primase
Nucleotides can ONLY be added to the 3’ end of
an already-attached nucleotide. A primer
provides this. DNA Primase builds the primer.
DNA
Polymerase Chain Reaction
• What does polymerase do?
• What if….
– I put an entire genome in a test tube.
– I added DNA polymerase III and dNTPs.
– I added very specific DNA primers that mapped
onto a specific gene.
– Then I started heating and cooling the DNA over
and over and over and over….
Polymerase Chain Reaction
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
5’
3’
-
taq polymerase
dNTPs
primers
buffer
Discuss:
• Find three similarities and three differences
between (i) DNA replication on the leading
strand and (ii) DNA replication via PCR.
(Debrief using name cards)
The 1st Challenge of Lagging Strand Replication
• DNA Primase is used over and over
• DNA Primase lays down ribonucleic acid.
Solution: an enzyme that can replace RNA
nucleotides with DNA nucleotides  DNA Pol1
The 2nd Challenge of Lagging Strand Replication
Solution: an enzyme that
can connect 3’ carbons to
phosphate groups 
LIGASE
5
Ends of parental
DNA strands
Leading strand
Lagging strand
3
3
5
Last fragment
Previous fragment
Lagging strand RNA primer
5
3
Parental strand
Removal of primers and
replacement with DNA
where a 3 end is available
DNA Pol1 Can only add nucleotides to the
3’ end of existing DNA.
5
3
Telomeres protect an organism’s
genes from being eroded during
successive rounds of DNA
replication. In humans the
sequence TTAGGG is repeated
between 100 and 1000 times.
Second round
of replication
5
New leading strand 3
New lagging strand 5
3
Further rounds
of replication
Shorter and shorter daughter molecules
Which of these replaces RNA with
DNA?
A)
B)
C)
D)
E)
DNA Polymerase 1
DNA Polymerase 3
DNA Primase
Ligase
Nuclease
Which of these glues 3’ and 5’ DNA
ends together?
A)
B)
C)
D)
E)
Ligase
DNA Polymerase 1
Binding Proteins
DNA Polymerase 3
DNA Primase
Which of these adds new nucleotides
to the leading strand?
A)
B)
C)
D)
E)
DNA Polymerase 3
The Sliding Clamp
DNA Polymerase 1
DNA Primase
Binding Proteins
Which of these is not needed by the
leading strand:
A)
B)
C)
D)
E)
DNA Polymerase 1
Binding Proteins
DNA Primase
The Sliding Clamp
DNA Polymerase 3
Which of these is not needed by the
lagging strand:
A)
B)
C)
D)
E)
The Sliding Clamp
DNA Polymerase 1
Binding Proteins
DNA Polymerase 3
All are needed
Why are telomeres needed?
a) Primers can’t bind to 5’ ends of DNA
b) DNA polymerase 1 can’t add nucleotides to 5’
ends of DNA
c) Okazaki fragments don’t form at the end of
DNA strands
d) Helicase can’t bind to the end of DNA
strands.
e) None of the above.
Write Down:
1. On a scale of 1 to 10, how well do you
understand DNA replication?
2. What is one thing you understand really
well?
3. What is one thing you do not understand?
Coming Up:
1.
2.
3.
4.
5.
Sanger Sequencing (Tues)
Molecular Sculpting of DNA replication (Tues)
Bonus assignment due (see Lecture Schedule)
Field trip sign up
DNA Replication Tutorial 3-5pm Friday