DNA replication requires four main components: DNA template, DNA polymerase, dNTPs, and magnesium ions. DNA unwinds and polymerase adds nucleotides based on complementary base pairing. DNA polymerase can only add to 3' ends so replication occurs in both directions from an origin of replication to yield two identical daughter DNA molecules. Topoisomerases are needed to resolve supercoiling that occurs during unwinding. Telomerase adds repeated sequences to chromosome ends during replication to compensate for incomplete end replication. DNA proofreading and mismatch repair help ensure high-fidelity copying of the genome.

1. DNA REPLICATION

2. Meselson and Stahl (1958)

3. 1958: Matthew Meselson & Frank Stahl’s Experiment
Semiconservative model of DNA replication

4. 1958: Matthew Meselson & Frank Stahl’s Experiment
Equilibrium density gradient centrifugation

5. Arthur Kornberg
Worked with E. coli.
Discovered the DNA POLYMERASE nd describe mechanisms of DNA synthesis.
Four components are required:
1. dNTPs: dATP, dTTP, dGTP, dCTP
(deoxyribonucleoside 5’-triphosphates)
(sugar-base + 3 phosphates)
2. DNA template
3. DNA polymerase (Kornberg enzyme)
4. Mg 2+ (optimizes DNA polymerase activity)
1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)

6. Model of DNA replication

7. Model of DNA replication

8. The Chemistry of DNA
replication

9. DNA Helicase
DNA double helix are tightly coupled. High
temperature is needed to break them (95oC)

11. DNA Synthesis by DNA polymerase

12. DNA Polymerase
Nucleotide polymerizing enzyme, first discovered in 1957

13. DNA replication Fork

14. DNA Binding Protein
SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins

15. SSB Proteins
DNA

16. DNA Clamping Protein

18. Cycle of DNA Polymerase/Clamping Protein loading and unloading
At the lagging strand..

19. Machinery for DNA replication

20. A Moving Replication

21. Structure of the Moving Complex

22. DNA winding

23. Topoisomerase
Opening the dsDNA will create supercoil
ahead of replication forks.
The supercoil constraint needs to be released
by topoisomerases.

25. The interconversion of topoisomers of dsDNA
is catalyzed by a topoisomerase in a three-
step process:
Cleavage of one or both strands of DNA
Passage of a segment of DNA through this
break
Resealing of the DNA break

26. Topoisomerase I (topo I)
Also called -protein in prokaryotes.
It cuts a phosphoester bond on one DNA strand, rotates
the broken DNA freely around the other strand to
relax the constraint, and reseals the cut.
Topoisomerase II (topo II)
It is named gyrase in prokaryotes.
It cuts phosphoester bonds on both strands of
dsDNA, releases the supercoil constraint,
and reforms the phosphoester bonds.

27. What about the ends (or telomeres) of linear chromosomes?
DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is
removed. this gap is not filled, chromosomes would become shorter each
round of replication!
Solution:
1. Eukaryotes have tandemly repeated sequences at the ends of their
chromosomes.
2. Telomerase (composed of protein and RNA complementary to the telomere
repeat) binds to the terminal telomere repeat and catalyzes the addition of of
new repeats.
3. Compensates by lengthening the chromosome.
4. Absence or mutation of telomerase activity results in chromosome shortening

28. Telomere replication

30. DNA Proofreading

31. Site-directed mismatch repair in eucaryotes
In DNAs are usually methylated on A while newly synthesized ones are not. So Cells can
distinguish old and newly synthesized DNAs and mutate mismatches on new ones.

33. Model of DNA replication

34. Model of DNA replication