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DNA Replication
C483 Spring 2013
1. Double stranded DNA that has been labeled with radioactive 14C is used as the
template for replication. Replication is carried out in a medium containing only
unlabeled nucleotides. After two rounds of replication, what percent of double
stranded DNA molecules are radioactive?
A) 25%
B) 50%
C) 75%
D) 100%
2. Which of these statements is false?
A) The primosome is part of the replisome
B) DNA polymerase I is part of the replisome
C) DNA polymerase III is part of the primosome
D) The Klenow Fragment is part of DNA Polymerase I
3. Which of these statements is false? DNA Polymerase III contains
A) 5’3’ exonuclease activity for proofreading
B) 5’3’ polymerase activity of elongation of the DNA strand.
C) Subunits to aid in processivity.
D) Subunits that act as “sliding clamps.”
4. Because the replisome synthesizes both strands of DNA simultaneously
A) Okazaki fragments are formed in the leading strand.
B) The leading strand is synthesized 5’3’, but the lagging strand is synthesized
3’5’
C) The replisome needs to have primase activity to make RNA primers for the
lagging strand
D) Proofreading activity is required for the leading strand, but not the lagging
strand.
5. Which of these activit(ies) is the last one in the process of lagging strand
synthesis?
A) Ligase
B) Polymerase
C) 5’3’ exonuclease
D) primase
History of Nucleic Acid Biochemistry
Miescher (1869)
Discovers nucleic acid
He called “nuclein”
Avery, MacLeod, McCarty (1944)
Kossel (1901)
Discovers five
nucleobases
A, G, C, T, U
Franklin and Wilkins
Watson and Crick (1953)
Semiconservative Replication
• Template
• Protein machinery
– DNA polymerase
• Three stages
– Initiation
– Elongation
– Termination
E. Coli chromosome
•
•
•
•
•
Circular, double stranded
Origin
Replication forks
Bidirectional
Replisome
– DNA polymerase is essential part
of the replisome
Bacterial DNA Polymerase II
• DNA Polymerase I and II
involved in repair and
polymerization of part
of one strand
• DNA Polymerase III is
major component of
replisome
• Multi-subunit,
asymmetric dimer
• Many activities
Polymerase Activity
• dNTP is substrate
• Added to 3’ end of growing
chain
– PRIMER is required
• 5’  3’ growth
• Selectivity based on Hbonding
– TEMPLATE is required
• Pyrophosphate generated
Processivity
• Replisome stays
attached to
replication fork
• Beta subunit is a
sliding clamp
• Higher efficiency
• Few replisomes
needed
Polymerase III Proofreads
• Incorrect base pair every
105 bp
• Epsilon subunit is
proofreader
• Unmatched base pairs have
different conformation
• 3’5’ exonuclease activity
• 99% of errors corrected
here
• 99% of remaining errors are
repaired
Polymerase Synthesizes Both Strands
Simultaneouly!
• Synthesis is 5’ 3’
• Leading Strand built on
the 3’5’ template
• Lagging Strand built on
the 5’3’ template
– Discontinuous
– Okazaki fragments
Primase
• RNA primer is needed
for each polymerization
• RNA primer is made by
primase enzyme
– Part of the primosome
complex
• Primosome, along with
DNA Polymerase III, is
part of replisome
Closing the Nick
• Three actions needed
• Removal of RNA primer
– DNA polymerase I
• Filling in the nick with
DNA
– DNA polymerase I
• Forming bond between
adjacent fragments
– DNA ligase
DNA Polymerase I
• Two activities similar to DNA polymerase III
– 5’3’ polymerase
• Extend chain
– 3’5’ exonuclease
• Proofreading
• Unique activity
– 5’3’ exonuclease
• Removes RNA primer
Klenow Fragment
• DNA polymerase I was
first discovered
– Kornberg Nobel prize
• Can be cut into two
pieces that retain
activity
• Klenow fragment
– Bigger piece with 5’3’
exonuclease removed
– “Usable” form of DNA
polymerase III
Three Step Process
Step 1
Step 2
Step 3
Mechanism of Ligase
Answers
1.
2.
3.
4.
5.
B
C
A
C
A