Quiz!
Reminders/Announcements
• Articles for projects are on the Toolkit website –
be sure you pick the project in which you can
attend the evening session.
• I will pass out guidelines for the paper.
• Start reading “The Double Helix”
• Wed Office Hours will be in the computer lab –
please help each other getting started
Review of Lecture 2
What type of molecule is this?
Name the three components.
What base is this?
is this a purine or a pyrimidine?
what would we have to change to make adenine?
What sugar is this?
what is the difference from the “other sugar”?
Give the full name of this molecule
Nucleic acids
• Polynucleotides linked 3' to 5' by phosphodiester bonds
• Ribonucleic acid (RNA) and deoxyribonucleic acid DNA
Summary of DNA Structure
5’
3’
3’
5’
Stabilizing interactions in DNA
Stabilized by:
• hydrogen bonds between
bases – “base pair”
• Stacking of bases through
van der Waals (π electronic
interactions and hydrophobic
interactions
• Hydrophobic effect- the
exclusion of water
The “canonical” base pairs
• The canonical A:T and G:C base pairs have nearly identical
overall dimensions – very important for the stacking in the helix
• A and T share two H-bonds
• G and C share three H-bonds
• G:C-rich regions of DNA are more stable
• Polar atoms in the sugar-phosphate backbone also form Hbonds
Watson – Crick Base Pairs
Canonical base pairs
Secondary Structure of DNA
• Sugar-phosphate backbone outside
• Bases (hydrogen-bonded) inside
• Right-twist closes the gaps between base pairs to 3.4 A
(0.34 nm) in B-DNA
Major and minor grooves
• The "tops" of the bases (as we draw them) line the "floor" of the
major groove
• The major groove is large enough to accommodate an alpha
helix from a protein
• Regulatory proteins (transcription factors) can recognize the
pattern of bases and H-bonding possibilities in the major groove
Helical and propeller twist in DNA
Comparison of DNA Structures
B-DNA
Right-handed
Major Groove – wide
Minor Groove – narrow
Pitch per turn helix – 33.2 Å
A-DNA
Right-handed
narrow
Broad
24.6 Å
Z-DNA
Left-handed
Flattened out on surface
Narrow
45.6 Å
Several Observed DNA Structures
Parameters used to describe the differences
between base pairs
Transition of B-DNA to A-DNA
Z-DNA
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•
•
•
Discovered by Alex Rich
Found in G:C-rich regions of DNA
G goes to syn conformation
C stays anti but whole C nucleoside (base and sugar) flips 180
degrees
Result is that G:C H-bonds can be preserved in the transition
from B-form to Z-form!
DNA Intercalators
Bis – intercalator
Minor and Major Groove Binding
Ligands and proteins bind to the major and minor grooves of DNA
Denaturation of DNA
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•
•
•
When DNA is heated to 80+ degrees Celsius, its UV absorbance increases
by 30-40%
This hyperchromic shift reflects the unwinding of the DNA double helix
Stacked base pairs in native DNA absorb less light
When T is lowered, the absorbance drops, reflecting the re-establishment of
stacking
Single stranded
Double stranded
Denaturation of DNA
reannealing
Denaturation of DNA – GC content
Tm
For long strands of DNA
Tm = 69o + 0.41(%G+C)
The three H-bonds in a G-C base pair
stabilize the DNA compared to the two Hbonds in an A-T base pair
Buoyant density of DNA is and index of GC content
η = 1.660 + 0.098 (mole fraction GC)
Tertiary Structure of DNA
• Length of E. coli DNA1.6 million nm,
but E. coli cell is only 2000 nm long
Therefore,
• DNA needs to be compacted and
folded
Supercoils – DNA tertiary structure
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•
In relaxed duplex DNA, ten bp
per turn of helix
Circular DNA sometimes has
more or less than 10 bp per
turn - a supercoiled state
L – linking number
T – twists
W – writhes
L=T+W
Overwinds DNA helix
relaxed
Unwinds DNA helix
supercoiled
Enzymes called topoisomerases or gyrases can introduce
or remove supercoils
Consider a fully relaxed circle of 400 bp double-stranded
circular DNA of linking number L = 40, twist T = 40 turns,
and writhe W = 0. By the action of topoisomerase, this
circle is supercoiled into a tertiary conformation of linking
number L = 36, with twist T = 40 turns.
The writhe of the resulting supercoil will be:
Is this negatively or positively supercoiled?
What is the consequence with respect to the DNA helix?
kDNA additional DNA tertiary structure
• Some bacteria and mitochondria (e.g. in
trypanosome) have concatenated DNA
Chromosome Structure - eukaryotes
• Human DNA’s total length
is ~2 meters!
• This must be packaged
into a nucleus that is about
5 micrometers in diameter
• This represents a
compression of more than
100,000!
• It is made possible by
wrapping the DNA around
protein spools called
nucleosomes and then
packing these in helical
filaments
What is the expected charge on a histone?
Chromatin is electron dense and composed of DNA and basic
histone proteins, which lie in the grooves of the double helix
DNA molecule. In regards to electrostatic interactions, what is
the expected charge on a histone?
Post translational modification of DNA -methylation
Mammalians cells only have 5methylcytosine (m5C) - ≈ 2 – 7%.
It is proposed that methylation of specific
cytosine residues regulates gene expression
Higher-order eukaryotes
methylated at adenines, N6-methyladenine
(m6A), in “GATC” sites
N4-methylcytosine (m4C).
5-methylcytosine (m5C)
Methylation regulates DNA replication as
well as protects DNA from DNA cleaving
enzymes
Prokaryotes
Bacterial m6A methylation – additional control of
replication
Methylation of the E. coli replication origin creates a refractory period for DNA
initiation. DNA methylation occurs at GATC sequences, 11 of which are found in the
origin of replication.
About 10 minutes after replication is initiated, the hemimethylated origins become fully
methylated by a DNA methylase enzyme.
The lag in methylation after the replication of GATC sequences is also used by the E.
coli mismatch proofreading system to distinguish the newly synthesized DNA strand
from the parental DNA strand; in that case, the relevant GATC sequences are scattered
throughout the chromosome.
Correlation with aberrant DNA
methylation and cancer
• Many tumor-suppressor and
other cancer-related genes
have been found to be
hypermethylated in human
cancer cells and primary
tumors
• The hypermethylation
silences the genes
DNA Damage
DNA Damage
Figure 5-46. A summary of spontaneous alterations likely to require DNA
repair. The sites on each nucleotide that are known to be modified by
spontaneous oxidative damage (red arrows), hydrolytic attack (blue arrows),
and uncontrolled methylation by the methyl group donor S-adenosylmethionine
(green arrows) are shown, with the width of each arrow indicating the relative
frequency of each event. (After T. Lindahl, Nature 362:709–715, 1993. ©
Macmillan Magazines Ltd.)
Deamination of Cytosine
Thyamine dimers
Nucleases
• Cleave nucleotide sequences
• DNases and RNases and non specific
nucleases
• ss and ds specificity
• Exonucleases (remove nucleotide from the
end)
• Endonucleases (recognize palindromic ds
DNA sequences)
Restriction endonucleases
• Three types (I, II, and III) – I and III require
ATP
• Type II are used as common molecular
biology tools
Type II restriction enzymes
• Recognize and cleave particular sequences
For example, BamHI
GGATCC
5’-N-N-N-N-G-G-A-T-C-C-N-N-N-N-3’
3’-N-N-N-N-C-C-T-A-G-G-N-N-N-N-5’
BamHI
5’-N-N-N-N-G-G-A-T-C-C-N-N-N-N-3’
3’-N-N-N-N-C-C-T-A-G-G-N-N-N-N-5’
5’-N-N-N-N-G
G-A-T-C-C-N-N-N-N-3’
3’-N-N-N-N-C-C-T-A-G
G-N-N-N-N-5’
“sticky ends” – overhanging sequence
Why do bacteria have
endonucleases?
How do they avoid digesting their
own DNA?
Overview
• DNA structure
– A, B, and Z DNA
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•
•
•
•
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DNA intercelators and groove binders
Thermal melting of DNA
DNA tertiary structure
DNA methylation
DNA damage
nucleases