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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