1. DNA Replication
C483 Spring 2013

2. 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?
The primosome is part of the replisome
DNA polymerase I is part of the replisome
DNA polymerase III is part of the primosome
The Klenow Fragment is part of DNA Polymerase I
3. Which of these statements is false? DNA Polymerase III contains
5’3’ exonuclease activity for proofreading
5’3’ polymerase activity of elongation of the DNA strand.
Subunits to aid in processivity.
Subunits that act as “sliding clamps.”

3. 4. Because the replisome synthesizes both strands of DNA simultaneously
Okazaki fragments are formed in the leading strand.
The leading strand is synthesized 5’3’, but the lagging strand is synthesized 3’5’
The replisome needs to have primase activity to make RNA primers for the lagging strand
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?
Ligase
Polymerase
5’3’ exonuclease
primase

4. History of Nucleic Acid Biochemistry
Miescher (1869)
Kossel (1901)
Discovers nucleic acid
He called “nuclein”
Discovers five
nucleobases
A, G, C, T, U
Franklin and Wilkins
Watson and Crick (1953)
Avery, MacLeod, McCarty (1944)

7. Semiconservative Replication
Template
Protein machinery
DNA polymerase
Three stages
Initiation
Elongation
Termination

8. E. Coli chromosome Circular, double stranded Origin Replication forks
Bidirectional
Replisome
DNA polymerase is essential part of the replisome

9. 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

11. Polymerase Activity dNTP is substrate Added to 3’ end of growing chain
PRIMER is required
5’  3’ growth
Selectivity based on H-bonding
TEMPLATE is required
Pyrophosphate generated

12. Processivity Replisome stays attached to replication fork
Beta subunit is a sliding clamp
Higher efficiency
Few replisomes needed

13. 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

14. 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

15. 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

16. Closing the Nick Three actions needed Removal of RNA primer
DNA polymerase I
Filling in the nick with DNA
Forming bond between adjacent fragments
DNA ligase

17. DNA Polymerase I Two activities similar to DNA polymerase III
Extend chain
3’5’ exonuclease
Proofreading
Unique activity
5’3’ exonuclease
Removes RNA primer

18. 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

19. Three Step Process

20. Step 1

21. Step 2

22. Step 3

23. Mechanism of Ligase

24. Answers B C A