Biochemistry I, Spring Term
Lecture 35
April 17, 2013
Lecture 35: DNA Stability/Protein-DNA Interactions, DNA Modification Enzymes
Key Terms:



DNA Stability
DNA-Protein Interactions
Restriction endonuclease & DNA ligase
1. Forces Stabilizing Nucleic Acid Structures.
Double stranded DNA & RNA can be reversibly
denatured ("melting"). Cooperative transition from
double stranded helix  single stranded random coils;
the change in absorbance of the bases at =260 nm can
be used to monitor this transition. The absorbance (A260) A260
increases when the DNA melts (hyperchromatic effect).
TM  %GC
Temperature
TM  [ NaCl ]
Tm
%GC or [NaCl]
Comparison of Dominate Forces in DNA and Protein Stability:
Energetic
Term
Protein
Stability
dsDNA
stability
Molecular Description of Energetic Terms in DNA stability.
H
H3C
H
Hydrogen
Bonds
+
++
O
N
H
N
N
H
N
N
N
N
O
O
O
O
Electrostatic
interactions
0
--
G
C
O
O
O P O
O
O
P O
O
O
O
T
A
O
O
O
O
CH3
Van der Waals
S
Conformational
Entropy
++
++++
++
-----
-----
--
--
N H
N
CH3
O
N
++++
+
O
N
N
O
1
O
H
H3C
Hydrophobic
Effect
O
N H
H
H
N
N
N
N
N
Biochemistry I, Spring Term
DNA Stability: The
two DNA molecules
on the left are mixed
under conditions that
promote formation of
double stranded
DNA. Draw any
forms of double
stranded (duplex)
DNA that would
form on the right.
Lecture 35
April 17, 2013
3’-A-C-G-T-5’
3’-A-T-G-C-A-G-A-C-G-T-G-5’
1 2 3 4 5 6 7 8 9 10 11
3’-A-T-G-C-A-G-A-C-G-T-G-5’
1 2 3 4 5 6 7 8 9 10 11
2. DNA-Protein
Interactions
Forces and functional groups involved in recognition.
1. Electrostatic bonding to the backbone.
G
a) side chains of Lys and Arg to
phosphates.
O
b) Release of metal ions (e.g.
K+) favors binding ( large
O
increase in ∆S of ions).
G
T
O
O
O
O
O
O P
T
O
O
O P
O
O
O
O
O
H3C
2. Van der Waals: Stacking (and intercalation) of Phe, Trp, and Tyr side
chains. More prevalent in single stranded (ss) nucleic acid.
ribose
O
N
N
H
O
H3C
3. Hydrophobic interaction with the 5-methyl of T.
N
ribose
O
NH
O
Thymine
Arg145
4. Non- Watson-Crick Hydrogen bonding to
the polar edges of the bases and to sugars.
a) Side chains of Arg, Asn, Gln, etc.
b) Protein main chain, C=O and NH
groups.
c) "Bridging H2O" can also participate.
N
O
N
H
H
H3C
O
O
N
O
N
O
O
2
NH2
Asn141
H
H
N
N
N
N
O
N
N
O
O
Biochemistry I, Spring Term
Lecture 35
April 17, 2013
Protein-DNA Binding: The following curves show the dissociation constants (KD) for DNA binding for three
different proteins (A, B, or C) as a function of salt concentration and DNA composition. Based on these
binding constants, which groups on the nucleic acid are recognized by which protein?
poly rU
10-9
O
Kd
O
O
10-8
P
O
H
O
A
O
O
N
10-8
10-7
3
Protein B: Binding very dependent on salt.
Protein C: Binding independent of salt  not phosphate.
Binding changes between rU and rC.
3. DNA Modifying Enzymes – ‘Cut and Paste’
B. Restriction Endonuclease: [endo - cut within,
nuclease - cleave nucleic acid]. Used by bacteria to
degrade invading viral DNA. Named after bacterial
species the particular enzyme was isolated from.
1. Enzyme binds to specific recognition sequences
with near absolute specificity and high affinity
(KD = 10-10 M).
2. Enzymes usually bind in major groove, forming
both specific and non-specific interactions.
3. Homodimeric enzymes have 180 degree
rotational symmetry. Because of the symmetry in
the enzyme, the DNA sequence also symmetrical.
The sequence is the same on the top and bottom
strands (referred to as palindromic sequences).
4. Require Mg2+ for cleavage, usually cleave both
strands at the same position. Generating a 3’OH.
O
O
O
G
O
O
O P
O
O
O
A
G
OH
O
O P
O
O
O
O
O
3
1
2
[NaCl]
Protein A: Binding independent of salt  not phosphate.
Binding same for U and C  not recognizing base.
A
H
O
3
N
N
O
O
O OH
C
2
[NaCl]
P
O
B
O OH
1
H
Kd
N
O
O
B
C
10-7
poly rC
10-9
A
N
H
Biochemistry I, Spring Term
Example restriction
enzyme cleavage
sequences are
shown on the right.
Lecture 35
Enzyme
April 17, 2013
Recognition
Sequence
Products
HindIII(A^AGCTT):-A-A-G-C-T-T-T-T-C-G-A-A-
-A
-T-T-C-G-A
A-G-C-T-TA-
BamHI(G^GATCC):
-G-G-A-T-C-C-C-C-T-A-G-G-
-G
-C-C-T-A-G
G-A-T-C-CG-
BglII(C^GATCG):
-C-G-A-T-C-G-G-C-T-A-G-C-
-C
-G-C-T-A-G
G-A-T-C-GC-
EcoRV(GAT^ATC):
-G-A-T-A-T-C-C-T-A-T-A-G-
-G-A-T
-C-T-A
HaeIII (GG^CC):
-G-G-C-C
-C-C-G-G
A-T-CT-A-G-
-G-G
-C-C
Example: EcoR1: G^AATTC
a) Non-specific interactions with DNA phosphates.
C-CG-G-
---G-A-A-T-T-C-----C-T-T-A-A-G-----G A-A-T-T-C-----C-T-T-A-A G-----G
---C-T-T-A-A
A-A-T-T-C--G---
b) Specific hydrogen bonds with donor and acceptors at the edge of bases in the major groove:
Arg145
G
NH2
Asn141
C
N
O
A
N
H
H3C
A
T O
O
N
O
T
C
H
N
H
H
N
O
A
N
N
O
O
G
minor groove
O
A-A-T-T-C-
-G A-A-T-T-C-
--G-A-A-T-T-C--
G-
-C-T-T-A-A G-
--C-T-T-A-A-G—
O
G
G
O
O
ATP
OH
Sticky end ligation:
-C-T-T-A-A
A
O
C. DNA Ligase – Uses ATP to join 5’phosphate to 3’-OH, provided the two groups
are in close proximity. Fragments created by the same restriction enzyme can always
be joined to each other.
-G
T
N
N
O
T
N
O
O P
O
O
O
A
ADP + Pi
O
O P
O
O
O
O
O
Blunt end ligation:
-G-A-T
-C-T-A
4
A-T-CT-A-G-
-G-A-T A-T-C-C-T-A T-A-G-
-G-A-T-A-T-C-C-T-A-T-A-G-
A