Download Class14 1-25 Win16 DNA Replication Notes

In Nature (2015):
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Wednesday, January 25th, 2017
Class 14 Learning Goals
DNA Replication
•  After this class, you should be able to:
–  Describe the genome-wide process of initiation of DNA replication
–  Define the role and predict a loss-of-function mutation result for
each of the enzymes involved in replication
–  Given a diagram of replicating DNA, locate likely sites of action for
each enzyme involved in replication
–  Assign descriptive terms appropriately to replication on the leading
or lagging strands of a particular replication fork
Polling Question #1
Which number is most likely the site of a
replication origin?
1
2
A chromosome
3
4
5
6
Peer Instruction
A chromosome being replicated
Circular chromosomes (like in prokaryotes)
Old
DNA
New
DNA
Origin of
replication
Linear chromosomes (like most eukaryotes)
3
5
3
5
3
What is an ‘origin’?
5
New DNA
3
5
Replication
bubble
Old DNA
Replication
fork
Why do eukaryotic chromosomes have multiple origins?
Why is replication able to go in both directions?
Today’s activity:
Each pair needs 1 packet.
a few polling questions will help us to
check in and keep on pace
2C: “Sketch” = basic parts (not a chemistry quiz)
Polling Question #2
Which of these enzymes is labeled incorrectly?
3) Primase
4) Topoisomerase
2) DNA Polymerase III
1) Ligase
Polling Question #4
Are these enzymes listed in the correct
order
of their operation on the lagging
5ʹ
3ʹ
strand for each Okazaki fragment?
3ʹ
5ʹ
5ʹ
3ʹ
Helicase,
5ʹ
then primase,
then DNA polymerase III,
4. DNA polymerase I removes ribonucleotides of primer,
then
DNA polymerase I,
1)  Yes
replacing them with dNTPs in the 5ʹ→ 3ʹ direction.
then ligase.
2)  No
5ʹ
3ʹ
5ʹ
Primase synthesizes
RNA primer
(supplying a 3’ OH)
5ʹ
3ʹ
5ʹ
Single-strand DNAbinding proteins (SSBP)
Peer Instruction
Topoisomerase
relieves twisting
forces
Helicase opens
double helix
Why can’t DNA replication start without a primer?
Why is ssBP important?
What would be the phenotype of a mutation in:
•  Topoisomerase
•  Helicase
Leading
strand
3ʹ
Peer Instruction
Sliding clamp holds
DNA polymerase III in place
5ʹ
RNA
primer
5ʹ
The sliding clamp has no effect on DNA pol’s ability to
catalyze one reaction. What does the sliding clamp do?
Why is this called the ‘leading’ strand?
DNA Replication Video
In which direction is:
•  the replication fork moving?
•  the polymerase on this strand moving?
Peer Instruction
What is the engineering problem faced by
the enzymes on the lagging strand?
RNA
primer
3ʹ
5ʹ
5ʹ
Topoisomerase
SSBPs
Primase
Helicase
Describe the mechanisms shown here.
5ʹ
3ʹ
5ʹ
Okazaki
fragment
Sliding
clamp
3ʹ
5ʹ
DNA polymerase
III
5ʹ
3ʹ
3ʹ
5ʹ
Okazaki
fragment
Okazaki
fragment
5ʹ
Peer Instruction
Polling Question #3
Because new dNTPs can only be replicated in the 5’ to 3’
direction, we know that the following statement must be
true about the strands of DNA created by DNA pol III:
1. 
2. 
3. 
4. 
5. 
Leading & lagging fragments are often different sizes
Leading & lagging fragments are often the same size
Leading moves more quickly than the lagging
Lagging moves more quickly than the leading
Single-stranded DNA-binding protein is not necessary
Peer Instruction
5ʹ
3ʹ
DNA polymerase I
3ʹ
5ʹ
5ʹ
What is DNA polymerase I doing?
5ʹ
3ʹ
DNA ligase
5ʹ
3ʹ
5ʹ
What would be the phenotype of a deleterious
mutation in the ligase-encoding gene?
Lagging Strand Video
Polling Question #5
Given what you know about DNA replication, which
of the following do you think is most likely?
1. 
2. 
3. 
More replication errors are created on the lagging strand
More replication errors are created on the leading strand
Neither strand ever creates replication errors during
replication
Homework: Replication Enzymes Chart
Enzyme
ssDBP
Topoisomerase
Helicase
DNA
Polymerase I
DNA
Polymerase III
Sliding Clamp
Primase
Ligase
Function Location Mutation
Effects?
Concept Questions
• 
How does replication begin on a single small linear chromosome? What proteins
are used?
–  How would this be different for an extremely large circular chromosome?
• 
Complete the given chart for the enzymes involved in replication. For each
enzyme, be able to justify the evolutionary advantage and protein cost of the
enzyme.
• 
Draw an upside down ‘Y’. Assume that the tail of the ‘Y’ is double stranded DNA.
Fill in the locations of all of the enzymes from the chart based on where they are
likely to act. You can assume that each arm of the ‘Y’ is 500 bases long and that
an Okazaki fragment is 150 bases long on average.
–  When finished, complete the replication bubble with the other fork.
• 
Which strand (leading or lagging) is best characterized as:
–  Simple?
–  Likely to have mutations?
–  More complicated in terms of enzymes
–  Likely to have a very long new strand
–  Bonus: Slower?
Peer Instruction
For years, you’ve heard that genes are things that are in your
DNA and can impact your life. What is a gene?
1) Does a gene include the coding region?
2) Does a gene include the non-template strand?
3) Does a gene include the stop codon?
4) Does a gene include the promoter?
5) Write a short, useful definition of a gene.
19
A working definition of the “gene” for Bio200:
A gene is a unit of genetic material that
encodes the information necessary to
produce one useful molecule.
–  Usually DNA
–  Often encoding a protein
–  Not necessarily continuous
–  Often guided by a promoter region