Chemical Biology: Scott Wolfe- Major Groove Recognition

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deoxythymidine

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deoxyadenosine

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deoxyguanosine

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deoxycytidine

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type of bond between ribose sugar and base

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glycosidic bond

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Base duplex formation mediated by:

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Watson-crick H-bonding between complementary bases (basepairing)

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draw a G-C base pairing

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Draw an A-T base pairing

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Which DNA surface (Major or minor groove) contains the most unique features for sequence specific DNA recognition?

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Major groove

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Major groove physical characteristics

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-wider and deeper
-most DBD recognition here
-Ave width: 11.6 A
-Ave depth: 8.5 A

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Minor groove physical characteristics

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-Ave width: 6.0 A
-Ave depth: 8.2 A

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which base pairing has more Propeller Twist?

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more propeller twist in AT vs. GC base pairs

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Some basepair parameters that affect the local conformation of the DNA

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-Rise: Ave is 3.3 A
-Helical twist: Ave is 36 degrees
-Propeller twist: ave is -11 degrees

-Many more parameters exist

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Local conformation of DNA is _____ dependent (KEY POINT)

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Sequence

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How do DNA-binding domains exploit DNA conformation?

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exploit sequence specific conformational flexibility/rigidity as well as local major/minor groove features to distinguish target sequence

-Example: AT regions usually have more compressed minor groove with hydration in minor groove

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Enthalpy in protein-DNA recognition

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favorable: H-bonds, electrostatic interactions, non-polar interactions (van der waals)

Unfavorable: desolvation of polar groups and induced structural strain (DNA or Protein)

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Entropy in protein-DNA recognition

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Favorable: Hydrophobic effect (release of ordered water form non-polar surfaces)

Unfavorable: reduced translational-rotational freedom, restricted vibrational motion, induced folding (protein)

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the energy of an electrostatic interaction is given by:

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Coulomb's law

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Hydrogen bond characteristics

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-H-bonds in water typically have energies of 1-3 kcal/mol
-H-bond lengths range from 1.5-2.6 A
-H-bonds are directional and polar

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van der Waals interactions

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-Arise from asymmetric distribution of electronic charge around an atom
-Induces complementary dipole in nearby atom=attractive force
-Interaction increases as distance decreases, until they are separated by van der Waals "contact"
-Short distances=strong repulsion
-VDW energies are small (.5-1 kcal/mol), but net effect over large molecules can be substantial

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EcoRI

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-Cleaves GAATTC site 50,000X more efficiently than single base mismatch
-funtions as dimer: one monomer recognizes surrounding sequence, one monomer cleaves

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Phosphodiester cleavage by Type II restriction enzymes

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-SN2-type mechanism
-Water is attacking nucleophile after deprotonation by neighboring phosphate
-Negative charge on attacked phosphate stabilized by Mg ion

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Equation representation of type II RE cleavage reaction

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Pentavalent TS
product has inverted stereochemistry
product= 5' monophosphate and 3' hydroxyl

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Method of studying the importance of specific contacts in a DBD

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-synthetic chemistry
-Use base substitution to investigate the impact of certain natural base contacts on the free energy of DNA binding

Ex: N6-methyl adenine, 7-deaza adenine, 2-deoxyuracil

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EcoRI base substitution study revealed:

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binding is cooperative, not additive

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Major rule for binding

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favorable energetic contributions of protein-DNA interaction largely offset by:

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entropic penalties associated with complex formation

-Genomic DNA will act as a sink to decrease free soluble protein concentration

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specific rules for base-specific DNA recognition by Zinc finger proteins?

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recognize adjacent DNA triplets

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No rules for DNA recognition, but there are trends:

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proteins take advantage of comlementarity between side chain donor/acceptor pairs and purine surfaces for recognition

-Arg likes N6 and O7 (usually G)
-Gln/Asn likes N6 exocyclic amines (usually A)

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Affinity does not = specificity

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Affinity is the difference in specificity between 2 different residues

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The balance between enthalpy and entropy in driving protein-DNA association

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Either Enthalpic or Entropic contributions can drive association

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TBP example

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TBP is rigid, and bends DNA to bind
-Enthalpically unfavorable
-Large hydrophobic surface is entropcically favorable, and this drives binding

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Some proteins bend DNA to bind; How is this achieved?

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At lease 2 different mechanisms: wedges or phosphate neutralization

-Over short distances (<150bp) bends are disfavored due to rigidity from base stacking interactions and coulombic repulsion of phosphate backbone
-TBP bends DNA by using "wedges": intercalating Phe side chains between neighboring base pairs
-CAP uses noncovalent interactions and selective phosphate backbone neutralization to induce DNA curve

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The challenge of site-specific DNA recognition

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Low protein concentrations (one individual protein) and high DNA concentration (per base pair) mean a ~4000 fold excess of non-specific DNA

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Challenge of sit-specific DNA recognition: funtion

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If the ratio of Ks/Kns is 10^3 and there are 4000-fold excess non-specific sites to specific sites, then 4-fold more protein will be bound to non-specific sites

-This problem increases with the size of the genome

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What problem does a large Ka^s present for a transcription factor?

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Half life is lengthened, so response times are too slow.

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How does a protein rapidly find it's preferred recognition sequence in the context of vast excess of non-specific sites?

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Many proteins form a non-specific initial complex with DNA based on electrostatics.

-A layer of water + weak electrostatics allows the protein to slide rapidly along DNA and hop between DNA segments= combination of 1D and 3D search for its target sequence

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Biological systems use cooperative binding to enhance specificity and the rate of equilibrium:

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steeper slope=faster response

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DNA damage recognition and repair

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-Repair enzymes must identify modified bases in a "sea" of standard DNA: must discriminate between modified and standard bases

-hOGG1 recognizes 8-oxo-guanine when paired with C in duplexDNA (This modified base can pair equally well with C or A during replication=G-C to T-A transversion)

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Base excision repair mechanisms

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1) hydrolysis to leave an Abasic site
2) Schiff base assisted hydrolysis and backbone cleavage

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One of the principle determinants exploited to recognize 8oxoG:

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change of the N7 position from a H-bond acceptor to a H-bond donor

-Also recognizes "orphan" C if paired with A

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Sequence specific recognition of the major groove can also be mediated by:

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A third strand of DNA (or RNA)
-binds major groove of DNA and recognizes the hoogsteen face of purine bases
-Binding can be parallel (hoogsteen) or antiparallel (reverse hoogsteen)to the strand that is being recognized
-This system provides a synthetic method for targeting desired DNA sequence

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purine-purine recognition using a third strand:

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requires antiparallel arrangement of interacting strands (reverse hoogsteen)