1. Genetics: DNA Replication

2. The relationship between structure and function is manifest in the double helix
Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material

3. 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end
Fig. 16-7a
5 end
Hydrogen bond
3 end
1 nm
3.4 nm
Figure 16.7 The double helix
3 end
0.34 nm
5 end
(a) Key features of DNA structure
(b) Partial chemical structure

4. Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand

5. Template or parental strands Daughter strands
Fig
Template or parental strands
Daughter strands A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C
(a) Parent molecule
(b) Separation of strands
(c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand
Figure 16.9 A model for DNA replication: the basic concept

6. DNA replication is rather remarkable….
Replication of chromosomal DNA occurs very rapidly
Very accurately
at the appropriate time in the life of the cell

7. Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model
They labeled the nucleotides of the old strands with a heavy isotope of nitrogen (N15), while any new nucleotides were labeled with a lighter isotope (N14).

8. Alternative methods of DNA replication
Fig
First replication
Second replication
Parent cell
The two parent strands rejoin
(a) Conservative model
Alternative methods of DNA replication
New strand contains one old and one new.
(b) Semiconserva- tive model
Figure Three alternative models of DNA replication
Each strand is a mix of old and new
(c) Dispersive model

9. Density centrifugation using a CsCl gradient
Fig
EXPERIMENT 1
Bacteria cultured in medium containing 15N 2
Bacteria transferred to medium containing 14N
RESULTS
Density centrifugation using a CsCl gradient 3
DNA sample centrifuged after 20 min (after first application) 4
DNA sample centrifuged after 40 min (after second replication)
Less dense
More dense
CONCLUSION
First replication
Second replication
Conservative model
Figure Does DNA replication follow the conservative, semiconservative, or dispersive model?
Semiconservative model
Dispersive model

11. What Meselson and Stahl did not know was how replication took place?
In 1960, Arthur Kornberg from Washington University, purified DNA polymerase from E.coli that was capable of synthesizing DNA.
Today we know that more than a dozen enzymes and other proteins participate in DNA replication

12. Getting Started
Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble”
Replication proceeds in both directions from each origin, until the entire molecule is copied.
The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication.

13. DNA replication always adds nucleotides onto the 3’OH of the growing strand. DNA synthesis is always 5’ to 3’ on the template strand.

14. Regulation of DNA replication (prokaryotes)
Specific proteins such as DnaA protein found in E.coli recognize DNA boxes within the origin (OriC in prok) and begin the replication.

15. Also a DAM protein methylates adenines to prompt regulation

16. In eukaryotes, much more complicated.

17. Difference in prokaryotic and eukaryotic DNA replication
A eukaryotic chromosome usually has many origins of replication.
The prokaryotic chromosome has one.

18. Parental (template) strand
Fig
Origin of replication
Parental (template) strand
Daughter (new) strand
Replication fork
Double-
stranded
DNA molecule
Replication bubble
0.5 µm
Two daughter DNA molecules
(a) Origins of replication in E. coli
Origin of replication
Double-stranded DNA molecule
Parental (template) strand
Daughter (new) strand
Figure Origins of replication in E. coli and eukaryotes
0.25 µm
Bubble
Replication fork
Two daughter DNA molecules
(b) Origins of replication in eukaryotes

19. Many enzymes are involved and are grouped together forming a DNA Replication Complex and the DNA winds through the complex like a reel in a movie projector.

20. At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating
Helicases are enzymes that untwist the double helix at the replication forks
Single-strand binding protein (SSB)binds to and stabilizes single-stranded DNA until it can be used as a template
Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

21. Single-strand binding proteins
Fig
Primase
Single-strand binding proteins 3
Topoisomerase 5 3
RNA primer
Figure Some of the proteins involved in the initiation of DNA replication 5 5 3
Helicase

22. The workhorses…..DNA polymerases
DNA polymerase is the enzyme(s) that catalyzes the formation of covalent bonds between adjacent nucleotides.
DNA polymerases III adds new bases.
DNA polymerase I removes RNA primers and fills in with DNA.
DNA polymerases II, IV, and V help in repair and replication of damaged DNA.

24. DNA polymerase III Is a large molecule made of
10 different subunits; the
complex is called DNA polymerase
III holoenzyme.

25. The polymerases are like hands.

26. DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end.
A short RNA primer is added initially. The 3 end of the RNA primer serves as the starting point for the new DNA strand.
An enzyme called primase adds the RNA primer according to base-pairing.
DNA polymerase I removes the RNA primers

27. RNA primers begin the process.
Fig
Primase
Single-strand binding proteins 3
Topoisomerase 5 3
RNA primer
Figure Some of the proteins involved in the initiation of DNA replication 5 5 3
Helicase
RNA primers begin the process.

28. Synthesizing a New DNA Strand
How fast is this?
The rate of elongation is about 750 nucleotides per second in bacteria and 50 per second in human cells
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

29. Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate dNTP (base, sugar, 3 phosphates)
As each nucleoside joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate.
This supplies the energy for the process.

30. Nucleoside triphosphate

31. Nucleoside triphosphate
Fig
New strand 5 end
Template strand 3 end
5 end
3 end
This is a
Source of
Energy for
DNA replication
Sugar A T A T
Base
Phosphate C G C G G C G C
DNA polymerase
3 end A T A T
Figure Incorporation of a nucleotide into a DNA strand
3 end C
Pyrophosphate C
Nucleoside triphosphate
5 end
5 end

32. Antiparallel Elongation
The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication
DNA polymerases add nucleotides only to the free 3end of a growing strand or to a primer; therefore, a new DNA strand can elongate only in the 5 to 3direction

33. Along one template strand of DNA, the DNA polymerase III synthesizes a leading strand continuously, moving toward the replication fork. A sliding-clamp protein moves DNA polymerase along the template strand.

34. Leading strands proceed toward the replication fork.
Fig a
Leading strands proceed toward the replication fork.
Overview
Origin of replication
Leading strand
Lagging strand
Primer
Lagging strand
Leading strand
Overall directions of replication
Figure Synthesis of the leading strand during DNA replication

35. a NEW DNA strand can elongate only in the 5 to 3direction 5
Fig b
DNA can only add at
the 3’ end of a growing
DNA strand or a primer.
Origin of replication 3 5
RNA primer 5
“Sliding clamp” 3 5
DNA pol III
Parental DNA 3 5
Figure Synthesis of the leading strand during DNA replication
a NEW DNA strand can elongate only in the 5 to 3direction 5 3 5

36. To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork
The lagging strand is synthesized as a series of segments called Okazaki fragments.
Another DNA polymerase (I) replaces the RNA primer with DNA nucleotides.
The fragments are joined together by DNA ligase.

37. Single-strand binding protein Overall directions of replication
Fig
Overview
Origin of replication
Leading strand
Lagging strand
Leading strand
Lagging strand
Single-strand binding protein
Overall directions of replication
Helicase
Leading strand 5
DNA pol III 3 3
Primer
Primase 5
Parental DNA 3
Figure A summary of bacterial DNA replication
DNA pol III
Lagging strand 5
DNA pol I
DNA ligase 4 3 5 3 2 1 3 5

38. Two DNA polymerase III proteins act in concert to replicate the leading and lagging strands. The term dimeric DNA polymerase is used to describe the two DNA polymerase holoenzymes that move as a unit toward the replication fork. For this to occur, the lagging strand is looped out with respect to the DNA polymerase that synthesizes the lagging strand. This loop allows the laggings-strand polymerase to make DNA in a 5’ to 3’ direction yet move toward the opening of the replication fork.

39. The DNA Replication Complex
Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules
HHMI's BioInteractive - DNA replication (advanced detail)
HHMI's BioInteractive - DNA replication (basic detail)

40. Fig. 16-UN5

41. DNA replication occurs with a high degree of fidelity.
H bonding between matched pairs is more stable than mismatched pairs.
The active site of DNA polymerase fits the correct base match with precision in an induced fit.
Enzymatic removal of mismatched nucleotides. Proof- reading….DNA polymerase removes mismatched or damaged nucleotides by exonucleases and replaces them.

42. Repair at the 3’ end

43. Telomeres in eukaryotic replication
Linear chromosomes contain telomeres at both ends
Telomeric sequences consist of a moderately repetitive tandem
Why are they needed? DNA polymerase is unable to replicate the 3’ ends of DNA strands so chromosomes would become progressively shorter.
Telomerase prevents chromosome shortening by synthesizing additional repeats of telomeric sequences.
Telomerase contains RNA and proteins. The RNA contains a complementary sequence to the DNA of the telomeric region and by a type of reverse transcription, replaces the DNA.

45. Can’t replicate here.

47. Unfortunately….
Telomerase is active only in germ cells, some types of stem cells such as embryonic stem cells, and certain white blood cells. And certain cancer cells

50. Summary of DNA replication in eukaryotic cells and prokaryotic cells
Prok – one replication bubble; Euk – many
Prok – circular chromosome; Euk- linear chromosomes with telomeres at ends
Prok – a few types of DNA polymerase; Euk – many types of DNA polymerases
Euk – more complicated, a lot we do not know about it for sure.

52. It looks like that the DNA was cut. It should have two bands, one is 1
It looks like that the DNA was cut. It should have two bands, one is 1.66kb and the other one is 4.1kb. There might be a weak band below or around the broken line of the gel on line 2 and 4 (it should be there). The uncut lines are correct (the supercoil DNA is the dominant form and run fast, the open circular is the weak band of slow running). I do not know why the ladder did not show up. We just used it and it was good. 
2016