lecture 6: dna binding interactionsn.ethz.ch/~nielssi/download/5. semester/biological chemistry i...
TRANSCRIPT
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Lecture 6: DNA Binding Interactions6.1 DNA‐DNA Interactions
– Antisense (plus modifications)
– Thermodynamics of DNA Duplex Formation
– Nucleobase Binding Sites
6.2 Protein‐DNA Interactions– Amino acid‐nucleic acid interactions
– Structures: Zn‐finger, helix‐turn‐helix, helix‐loop‐helix, leucine zipper
6.3 Small molecule‐DNA Interactions and modifications– Sequence‐Specific Binding by Small molecules (Polyamide code‐
reading)
– DNA intercalation
– Overview of Nucleic Acid Modifications
– DNA oxidation and photodamage
ANTISENSE OLIGOS
Crooke ST, Annu. Rev. Med. 55, 61-95 (2004)
(1)single‐strand DNA oligopasses through cell membrane and enters cytoplasm.
(2)oligo enters nucleus. (3)oligonucleotide binds
(hybridizes) to the target mRNA, forming a sense‐antisense duplex.
(4)formation of the duplex initiates the recruitment of the RNase H enzyme, an endogenous nuclease.
(5)RNase H degrades the target mRNA, inhibiting target mRNA expression.
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Nucleic Acid‐Nucleic Acid
Protein‐Nucleic Acid
Small Molecule‐Nucleic Acid
Grooves, Backbone, and Places in Between
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H‐BONDING IN THE DNA GROOVES
Amino Acid‐DNA Binding Interaction
Some residues commonly used for base‐specific contacts:AsparagineGlutamineArginineHistidineTryptophan
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TRANSCRIPTION FACTOR FAMILY ALBUM
Luscombe et al., Genome Biol. 1, 1-37 (2000)
Homeodomain Lac Repressor Zn Finger Gal4HELIX-TURN-HELIX ZINC BINDING
ZIPPER-TYPE PROTEINSLeucine zipper Helix-loop-helix
ß-SHEET PROTEINSMet repressor NF-B
ZINC FINGER PROTEINS
S.A. Wolfe et al. Annu Rev. Biophys. Biomol. Struct, 29, 183-212 (2000)
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DNA BINDING BY ZN FINGER PROTEINS
HELIX‐TURN‐HELIX PROTEINS: 1
Red (3): DNA recognition helix
Blue (2) and Green (1): responsible for positioning the recognition helix in the major groove of DNA
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Helix‐turn‐helix proteins: 2
This Homeodomain protein is another Helix-Turn-Helix protein
It contains a N-terminal extension on Helix 1 that binds in the minor groove of DNA
LEUCINE ZIPPER PROTEINS
Dimerization domain: Coiled-coil (leucine zipper)
DNA binding domain: Two -helicses, positioned by dimerization domain
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HELIX‐LOOP‐HELIX PROTEINS
Dimerization domain: Four helix bundle
DNA binding domain: Two -helicses, positioned by four helix bundle
deaminases can “edit” nucleic acids. For example: ADA catalyzes this transformation:
Consider Enzyme X that deaminates deoxyadenosine. The Kd for Enzyme X is higher for a duplex with 7‐deaza‐A:T than for an A:T
Propose a rationale for this observation.
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6.3 SMALL MOLECULE‐DNA INTERACTIONS AND MODIFICATIONS
RECOGNITION OF DNA IN THE MINOR GROOVE
• In one turn of the double helix (10 bp) all possible combinations of the four watson‐crick base pairs afford 524,800 different sequences
• Can small combinations of small molecules formulate the basis of a chemical code for recognizing linear combinations of Watson‐Crick pairs?
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NETROPSIN‐DNA COMPLEX
DISTAMYCIN‐DNA COMPLEX
• Distamycin A enhances site‐specific double‐strand DNA cleavage
• Hypothesis: DNA conformational change by binding allows cleaving agent to interact at specific sites
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RECOGNITION OF THE MINOR GROOVE BY DIMERIC POLYAMIDES
Dervan PB, Bioorg. Med. Chem. 9, 2215-2235 (2002)
DISCRIMINATION BETWEEN A‐T AND T‐A
Dervan PB, Bioorg. Med. Chem. 9, 2215-2235 (2002)
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RULES FOR THE RECOGNITION OF DNA BY POLYAMIDES
Dervan PB, Bioorg. Med. Chem. 9, 2215-2235 (2001)
DOXORUBICIN (Adriamycin®) ‐ A DNA INTERCALATOR
The flat B-C-D rings intercalate in the DNA and this binding is stabilized by the sugar moiety of the molecule that contains the amino group.
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N
NN
N
O
DNANH2
CH3
N
NN
N
NH2
DNA CH3
N
NHN
N
O
DNANH2
H3C
N
N
DNA
O
NH2
H3C
7-Mehtylgunanine (G)relatively harmlesssubstrate for Aag
3-Mehtyladenine (A)replication blocksubstrate for Aag
O6-Mehtylguanine (G)replication blockmutagenic (G->A)substrate fŸr Agt
N3-Mehtylcytosine (C)replication blocksubstrate for AlkB
FREQUENT SITES OF METHYLATION IN DNA
Photoproduct Induced Double Helix Distortion
Rastogi, R. et al. J. Nuc. Acids. 2010. 2010, 32
CPD (6‐4) PP (6‐4) Dewar Dimer
Large distortion of the DNA backbone