1. DNA Structure

2. DNA Backbone alternating sugar-phosphate backbone
sugar and phosphate backbone attached by a phosphodiester bond
DNA is said to be read from 5’ to 3’
opposing backbones are antiparallel 5’ 3’ 1’ 2’ 4’
Antiparallel = upside down
5’  3’ labeled similarly to proteins with numbered carbons (α1-4), except we use prime for DNA.

3. Nitrogenous Bases

4. Complementary Base Pairing
Chargaff’s Rule
adenine : thymine (1:1)
guanine : cytosine (1:1)
the diameter of a base pair is 2 nm
3 H-bonds between G&C
2 H-bonds between A&T
2 nm = 2 nanometers

5. Overall DNA Structure right handed helix
0.34 nm between adjacent base pairs
one turn of the helix is 10 base pairs (3.4 nm)
A nm = one thousand-millionth of a m. It can be written as 1 m / 1,000,000,000.
0.34 nm
3.4 nm

6. Overall DNA Structure minor groove major groove
Major and Mionor groove because DNA molecule is not a perfect helix. Consider a spiral stair case What makes it uniform in diameter? The support beam down the centre. There is no “support beam” down the centre of a DNA molecule.

7. Three DNA Types
A-DNA
B-DNA
Z-DNA

8. Three DNA Types 1 angstrom = 1.0 × 10-10 meters
A-DNA
B-DNA
Z-DNA
helical rotation
right-handed
left-handed
# base pairs per 360°
10.7 10 12
helical diameter
25.5 Å
23.7 Å
18.4 Å
major/minor grooves
yes
no – all grooves very similar in height
Don’t need to memorize.
A and B DNA are found naturally in our bodies.
Z DNA reverse direction, or left handed. Much less common.

9. DNA Structure - Review

10. DNA Replication

11. DNA Replication
All cells are capable of giving rise to a new generation of cells through DNA replication (copying) and cell division.
Mitosis: replicated DNA in nucleus is divided equally between two daughter cells

12. 1958 – Meselson & Stahl http://www.youtube.com/watch?v=JcUQ_TZCG0w
grew E. coli in 15N media
14N is the usual isotope; 15N is heavier
ensured that the bacterial DNA only contained 15N

13. Dark Blue = parental strand (original DNA)
Figure a–c
The two
parental strands
reassociate
after acting as
templates for
new strands,
thus restoring
the parental
double helix.
strands of the
parental molecule
separate,
and each
functions
as a template
for synthesis of
a new, comple-
mentary strand.
Each
strand of both
daughter mol-
ecules contains
a mixture of
old and newly
synthesized
DNA.
Parent cell
First
replication
Second
CONSERVATIVE MODEL
Dark Blue = parental strand (original DNA)
Light Blue = new strand
SEMI-CONSERVATIVE MODEL
DISPERSIVE MODEL

14. Meselson & Stahl Experiment
DNA was isolated and its weight determined through centrifugation

15. DNA Replication DNA replicates in a semi-conservative manner
one strand of DNA is used as a template for the other strand
5’ A T G T C A G 3’
3’ T A C A G T C 5’

16. 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 5’ 3’
(a) The parent molecule has two complementary strands of DNA Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.
(b) The first step in replication is separation of the two DNA strands.
(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.
(d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands Each “daughter” DNA molecule consists of one parental strand and one new strand. A C T G
Figure 16.9 a–d 5’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’

17. DNA Replication
DNA replication starts at a number of sites along a chromosome – origins of replication
dozens of proteins are involved in the process of DNA replication

18. DNA Replication Initiation
DNA helicase – unwinds DNA to break its bonds
single-stranded binding proteins (SSBPs) – bind to each of the single stranded pieces of DNA and keeps them seperated
if there were nothing to keep the strands apart, they would reanneal (stick back together)
DNA Replication Animation

19. Proteins / Enzymes of DNA Replication
as DNA is being unwound by helicase, DNA in front of the helicase gets bunched up
DNA gyrase – enzyme that loosens the tension in front of the replication fork
DNA gyrase – shoe lace demo (prevents coil from unwinding too much or getting too tight)
DNA polymerase III – main enzyme of DNA replication, called III simply because it was discovered after DNA polymerase I.
RNA primer – needed to start relipcation 19

20. Replication Structures
replication occurs in both directions from the origin of replication
replication fork – structure generated by DNA helicase and SSBPs
replication bubble – two replication forks which are close in proximity

21. DNA Replication Part 2

22. DNA Polymerase
DNA polymerase – enzyme which synthesizes nucleotide chains
in prokaryotes: DNA polymerase I, II, III, IV & V
in eukaryotes: over 15 different types
nucleotide chains only form in the 5’  3’ directionn (can only add to the 3’ end of the new strand)
DNA polymerase III is primarily responsible for DNA replication in prokaryotes

23. DNA Polymerase III Substrates
DNA template
deoxyribonucleoside triphosphates (dNTPs)
RNA primer 3’ 5’ 5’ 3’ 3’ 5’

24. ATP (Adenosine Triphosphate)
ADENINE
3 PHOSPHATES
RIBOSE

25. 3’ Figure 16.13 New strand Template strand 5 end 3 end Sugar A T
Base C G P OH
Pyrophosphate
2 P
Phosphate 3’
DNA Polymerase
deoxyribonucleoside triphosphates (dNTPs)

26. Step 1
Primase makes a short RNA primer on the exposed single-stranded DNA (ssDNA). 5’ 3’ 3’ 5’

27. Steps 2, 3, and 4
DNA polymerase III binds to the end of the RNA primer.
The appropriate nucleoside triphosphate binds to the polymerase.
A pyrophosphate group (PPi, a diphosphate group originally isolated by heating phosphates) is cleaved, while the nucleotide is added to the end of the nucleotide chain.
TTP P P T A A

28. 3’ Figure 16.13 New strand Template strand 5 end 3 end Sugar A T
Base C G P OH
Pyrophosphate
2 P
Phosphate 3’
DNA Polymerase
deoxyribonucleoside triphosphates (dNTPs)

29. Leading Strand
DNA polymerase III 3’
SSBPs 5’ 3’
primase
gyrase
helicase 5’ 3’
The leading strand is synthesized continuously during DNA replication 5’

30. Lagging Strand 5’ 3’ 3’ 5’ 3’
Okazaki fragment 5’

31. Connecting Lagging Strands
DNA polymerase I – removes the RNA primer
occurs in the 5’  3’ direction
DNA ligase – connects the sugar-phosphate backbone (by creating phosphodiester bonds) of Okazaki fragments
Okazaki fragments are typically 1000 to 2000 nucleotides (NTs) in length

32. Replication Animation
DNA replication occurs:
continuously on the leading strand
discontinuously on the lagging strand
Replication Animation

33. Replication Overview - 1
helicase unwinds the double stranded DNA structure creating a replication fork
the single stranded region of the replication fork are maintained by SSBPs
gyrase relieves the tension ahead of the replication fork

34. Replication Overview - 2
two original parent strands serve as templates for the new daughter strands
daughter strands are produced in one of two methods
leading strand (continuous polymerization)
lagging strand (discrete polymerization)
1000 – 2000 NTs Okazaki fragments joined together

35. Replication Overview - 3
primase begins each new daughter strand with a short RNA primer
DNA polymerase III extends a DNA strand from the RNA primer
DNA polymerase I removes the RNA primer AND fills it in with DNA
DNA ligase joins the sugar-phosphate backbones of all adjacent DNA segments

36. Mistakes in DNA Replication
less than 1 error in 107 (10 million) NTs
exonuclease – enzymes which can cut out sections of nucleic acids
DNA polymerase I and III have polymerase and exonuclease activity to fix mistakes

37. DNA Repair Figure 16.17 A thymine dimer distorts the DNA molecule. 1
A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed. 2
Nuclease
DNA
polymerase 3
Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
ligase
DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete. 4
Figure 16.17

38. Homework helps prepare you for tests!!!!
Homework will be checked each class:
Pg. pg. 209 #1-4
pg. 216 #1-9
Pg. 223 #1-5, 7-8