Biochemistry 330

Nucleotide Structure

* Phosphate
* Base (A,T,G,C,U)
* 5 Carbon Sugar (Ribose or Deoxyribose)

\
* Covalent Bonds:
* Glycosidic Bonds
* Phosphodiester Linkages

Nucleoside

* Base (A,T,G,C,U)
* 5 Carbon Sugar (Ribose or Deoxyribose)

Purine

* Adenine & Guanine
* 9 Bi-Cyclic Structure

Pyrimidine

* Thymine, Uracil, Cytosine
* 6 Cyclic Structure

Adenine

* Purine
* NH2 Only

Guanine

* Purine
* NH2
* Carbonyl

Thymine

* Pyrimidine
* 2 Carbonyl
* Methyl Group

Uracil

* Pyrimidine
* 2 Carbonyl

Cytosine

* Pyrimidine
* 1 Carbonyl

Where does Phosphate Attach on Nucleotides?

5’ Carbon of Pentose Sugar

Where are Pyrimidine Bases and Sugars Bonded?

N1 of Base attached to 1’ C of Sugar

Where are Purine Bases and Sugars Bonded?

N9 of Base attached to 1’ C of Sugar

Diphosphate

2 Phosphate attached to 1 Carbon

Biphosphate

2 Phosphate and 2 Carbon

Nucleic Acid Structural Hierarchy

__**Primary**__

* Covalent Bonds (Glycosidic within 1 nucleotide)
* Phosphodiester Bonds (between nucleotides)
* Asymmetric Linkages
* Creates 3’ to 5’ Directionality
* Overall (-) Charge
* Sequence

\
__**Secondary**__

* Helical Structure
* B-DNA, A-DNA, Z-DNA
* Base Pairing
* Hydrogen Bonding
* Base Stacking Interactions
* Hydrophobic Interactions
* Ionic

\
Tertiary (Higher Order)

* Folded Nucleic Acids
* tRNA, rRNA

What Causes Directionality in Nucleic Acids?

* Asymmetric linkages of phosphodiester bonds
* Causes 3’-5’ Direction
* Causes overall (-) Charge

Direction to Read Sequences?

5’ to 3’ Direction

Chargaffs Rule

A=T

G=C

Tautomers

Isomers that differ only by the location of protons

* Keto favoured over Enol

\
* Can impact replication/mutations

B-DNA

* Regular/Hydrated DNA
* 10-10.5 Base Pairs/Turn
* Major & Minor Groove
* Anti-Parallel
* 5’ - 3’
* Right Handed

Central Dogma

* Once converted into proteins, primary structure information cannot be converted into nucleic acids

Major & Minor Groove

* Major Groove: Backbone Far Apart
* Minor Groove: Backbone Close Together
* Both grooves can have Hydrogen bonding beyond base-pairs

A-DNA

* Irregular/Dehydrated DNA
* Right Handed
* Wider Minor Groove
* Narrow Major Groove
* 11.6 Base Pairs/Turn
* Sugar Conformation: C3’ Endo

Sugar Conformation

Plane Determined By: C1’, C4’, O

* Above Plane: ***endo***
* 3’ Endo : Phosphate ***close*** together
* 2’ Endo : Phosphate ***far*** apart
* Below Plane: ***exo***

\
B-DNA: 2’ Endo

A-DNA: 3’ Endo

Z-DNA

* High Salt
* Alternating Purine/Pyrimidine (GCGCGCGC)
* Left-Handed
* 12 Base-Pairs/Turn
* Minor Groove Only
* Purines: Syn Conformation
* Pyrimidines: Anti Conformation

Anti (Base Conformation)

* Preferred Base Conformation
* Places bulky groups further from sugars
* Reduces steric strain

\

3’ Endo (Sugar Conformation)

* A-DNA
* Phosphate Closer Together
* 3’C and 5’C in ***same plane***

2’ Endo (Sugar Confromation)

* B-DNA
* Phosphate Farther Apart
* 2’C and 5’C in ***different plane***

Syn (Base Conformation)

* Base conformation for purines in Z-DNA
* Brings base above sugar

Endonucleases

* Cut DNA of interior sequences
* \

Exonucleases

* Cut terminal nucleotides

Restriction Endonucleases

* Cut/Break DNA in a specific sequence
* Used by bacteria to degrade foreign DNA
* Base modification (methylation) to protect bacterial genome
* Target Palindromic DNA

\

Palindromic DNA

Sequence of strands matches

EcoRI

* G|AATTC (Palindrome)
* 5’ Overhang
* Interaction is in the ***major groove***

EcoRV

* GAT|ATC (Palindrome)
* Blunt (No Overhang)

Isoschizomers

2 Enzymes cleaving the same sequence

Restriction Endonuclease (Type IIP)

* Type of Restriction Endonuclease
* Palindromic
* Cuts within recognition sequence

Restriction Endonuclease (Type IIS)

* Type of Restriction Endonuclease
* Non-Palindromic/Staggered
* Cuts outside/adjacent of recognition sequence
* Ends create more variability

3’ Overhang

EXTRA BASES on 3’ end

5’ Overhand

EXTRA BASES on 5’ end

What Protects The Bacterial Genome from Restriction Endonucleases?

* Base modifications (Methylation)

Electrophoresis

* DNA Purification Method
* Gel Matrix (Agarose/Cross-Linked Polyacrylamide)
* Higher % Agarose/Polyacrylamide ***increases resistance***

\
* Separations can distinguish between single nucleotide differences in size
* Top/Slower: Heavier, longer chain, bent, ***relaxed***
* Bottom/Fast: Lighter, shorter chain, straight, ***supercoiled***

\
* DNA travels through an electric field
* Carried negative charge with a consistent charge/mass ratio
* \

\
Visualization After

* UV light with appropriate dye (Ethidium Bromide)

\

Linking Number (L)

* Number of times each strand twists around the other
* Wont change unless covalent bonds are broken

\
* L = T + W

Twist (T)

* Always equals Lk (Linking number at rest)

\
* Twist = #Base Pairs/#Base Pairs Per turn
* B-DNA: 10-10.5 BP/turn
* A-DNA: 11.6 BP/turn
* Z-DNA: 12 BP/turn

Writhe (W)

* Can be +/-
* Depends on handedness of DNA

Topoisomerases

* Enzyme
* Changes linking number by breaking phosphodiester backbone (covalent bonds)

\
* Type I
* Breaks 1 Strand

\
* Type 2
* Breaks 2 Strands
* Requires ATP

Type IA Topoisomerase

* Linking Number: +1
* Break one strand and pass the other “through” the break

\
* Tyrosine enzyme covalently attached by a 5’ phosphate linkage
* Energy from bond transferred to new bond to enyzme

Topoisomerase III (E.coli)

* Type IA Topoisomerase
* Linking Number: +1
* Single Polypeptide
* 4 Domains

\
* Has ***Large Openings***
* (+) Charge Residues: Arginine/Lysine
* Accommodates duplex DNA structures

Type IB Topoisomerase

* Linking Number: +/-1
* Writhe decreases in magnitude (relaxes both +/- supercoils)

\
* Tyrosine enzyme attaches to a 3’ phosphate
* Once covalent complex is formed, non-covalently attached portion ***rotates to relieve supercoiling***

Topoisomerase I (E.coli)

* Un-cleaved Portion of DNA

\
* Cleaved Portion of DNA
* Upstream
* Covalently attached to Tyrosine
* Downstream
* Non-covalently associated with ***positive amino acids (Arginine/Lysine)***
* Attachments ***not in fixed orientation***

\
* DNA is released one phosphodiester bond is re-formed

Type II Topoisomerase

* Linking Number = x2

\
* ATP dependent
* Breaks covalent bonds of double-strands

DNA Gyrase

* Prokaryotic Topoisomerase II

\
* Increase (-) supercoiling
* Decreases Linking number
* Writhe: -2/cycle

\
* Tyrosine forms bond to 5’end of cleaved strands with a 5’ sticky overhang

Topoisomerase II (S. cerevisiae)

* Homodimer
* ***DNA is*** ***deformed*** in association with protein
* Protein interacts with backbone ***(not specific)***

Where are Topoisomerases Important?

* Important in replication and transcription

\
* Inhibiting topoisomerases can be useful for cancer treatment
* Topoisomerase inhibitors stop replication from occurring
* Cancer cells stop multiplying

What Forces Stabilize Secondary Structures?

Non-covalent Forces

* Hydrogen bonds
* Watson-Crick Base Pairs
* Other Pairing Interactions (Hoogsteen Pairs)
* Bases that cannot form hydrogen bonds are ***destabilizing***

\
* Base Stacking Interactions
* Hydrophobic Interactions
* Van der Waals
* Can occur even in absence of duplex
* GC pairs have ***stronger stacking interactions*** than AT

DNA Denaturation

* Double Strand → Single Strand

\
* Occurs in response to:
* Increased Temperatures

\
Results in:

* Lower viscosity (thicker/less fluid) solutions
* Higher Absorbance (UV Light)

Hyperchromicity

* 40% increase in UV absorbance at 260nm
* Increase in absorbance as bases become less ordered
* Native → Denatured

Transition Midpoint (Tm)

* Melting Point of DNA
* Double → Single Stranded
* Cooperative Process

\
* Indicator of Stability
* Increases with higher GC content
* Decreases with higher T content

High GC Content

* Increase in Transition Midpoint (Tm)

Higher T Content

* Decrease in Transition Midpoint (Tm)

Renaturation

* Single Strand → Double Strand
* Can occur ***depending on complexity of DNA***

\
* **Highly Repeated DNA:** Rapid

\
* **Moderately Repeated DNA:** Intermediate

\
* **Unique DNA:** Slow

RNA

* Similar to DNA structure but ***structures are more varied***

\
Mixtures of…Within a Single Strand:

* Helical
* Single Stranded

\

What Forces Stabilize RNA Structures?

* For RNA

\
* Stacking Interactions

\
* Hydrogen Bonds
* Watson-Crick
* Non-Watson Crick
* Ex: GU pairs

Non-Specific Protein Binding to DNA

* Type of Protein Binding to DNA

\
* Interactions will be to backbone
* Ionic and Hydrogen Bonding Interactions

\
* Ex: Histones/Nucleosomes
* Can distort DNA (induce bending)

Specific Binding to DNA

* Type of Protein Binding to DNA

\
* Interactions will be with bases or sequence-specific structures

\
* Ex: Restriction Endonucleases

DNA Binding Motifs

* Zinc Fingers
* Coiled-Coils
* Helix-turn-helix
* **Interactions:** Alpha-helix binding into major groove

Nucleosomes

* Compact DNA in Eukaryotes
* Mixture of DNA & Protein (Chromatin)
* Histones are major protein component in chromatin

\
* DNA winds around it \~1.65 times
* Left-handed winding
* Introduced overall (-) supercoils

\
* Core: Equal amounts of
* H2A, H2B, H3 and H4 (2 of each, octomer)

\
* ***Non-Specific Interactions*** between Backbone and positively charged amino acids

\
\
* Pack together to form a 30nm fibre

Histones

* Important in first steps of DNA condensation into nucleosomes

\
* Highly Conserved
* Indicates important structural/functional role
* Serine (S)/Threonine (T)
* Isoleucine (I)/Valine (V)
* Arginine (R)/Lysine (K)

\
* Small (100-215 Amino Acids)
* Can be modified post-translationally (methylation, acetylation, phosphorylation)
* Positively Charges (\~20-30% Arg, Lys Residues)
* H1, H2A, H2B, H3, H4

Chromatin

* Contains Histones
* Equal Amounts of: H2A, H2B, H3, and H4

Histone H1

* Part of intact nucleosome (released during nuclease treatment)
* 2 full turns of DNA

30nm Fibre

* Created as a result of nucleosomes packing together

\
* Stabilized by:
* Core Histones: H2A, H3, and H4

\
* Can further pack into higher-order ***nuclear scaffold***
* "Loops” and “Rosettes”

What is Critical for Higher-Order Structures?

* Topoisomerase are critical
* To generate nucleosomes and higher

Higher Order Structures

Histone + DNA → Nucleosome → 30nm Fibre → Scaffold (Loops/Rosettes)

Conservative Replication

* Form of Replication
* Strands remain intact
* Parent structure remains intact
* Daughter strands are newly synthesized

Semi-Conservative Replication

* Form of Replication
* Strands remain intact
* Parent structures are hybrid with new strands (1:1 ratio)

Dispersive Replication

* Form of Replication
* Strands ***do not remain*** intact
* New synthesis has both old and new portions distributed evenly

Direction Which Replication Proceeds?

* 5’ → 3’
* 3’OH of strand is ***nucleophile***
* a-phosphate of a nucleotide triphosphate is the ***electrophile***

Where Does Replication Occur?

* Occurs at the replication fork
* Progressive labelling with radioactive isotopes shows location and direction of incorporation of new bases

Replication Bubble

* Characterized 2 oppositely-moving replication forks

Semi-Discontinuous

* 1 Replication Bubble


* 4 strands are being synthesized at the same time

\
* As one strand is being continuously synthesized, another stops continuously (lagging strand: Te

Lagging Strand

* 5’-3’ Template Strand
* Results in Okazaki fragments

What Attack Occurs as Nucleotides are Added?

* Nucleophilic Attack
* Nucleophile: 3’OH Strand
* Electrophile: Phosphate of nucleotide triphosphate

\
* Assisted by Polymerase
* A Metalloenzyme (has Mg2+)

Replication Process

1. Origin of Replication

* Has high AT content
* Template strand is hemimethylated

\

2. DnaA binds to AT region

* Causes initial unwinding at AT-rich regions
* **ATP hydrolysis**

\

3. Helicase DnaB binds to ***lagging strand*** (Template: 5’-3’)

* Unwinds strands
* Single strands are now exposed

\
* Single Stranded Binding Protein (SSBP)


* Prevents:
* Reannealation
* Formation of secondary structures
* Endonuclease effects

\

4. DnaG (Primase) binds to lagging strand and leading strand

* Synthesizes initial RNA primers without the need for a complementary strand

\

5. Polymerase III

* Adds complementary nucleotides on both strands simultaneously (1000 nucleotides/second)
* Lagging strand synthesized in a loop
* Also does proofreading

\
* Polymerase III processivity can be increased with the presence of ***Sliding clamps (beta-clamps)***

\

6. Polymerase I

* Removes the RNA primers

\

7. Dna Ligase

* Glues Okazaki fragments together
* ***Can use ATP or NAD+ (Depending which kind of Ligase)***

\

8. Once all are assembled (replisome) Ends are fully-methylated

* DnaA can only bind to fully methylated strands
* Therefor methylation allows for replication to only occur once per cell division

\

9. Once arrived at Ter sequence, replication stops

* TerA/TerC are most common termination sites

\

10. Two interwound ***(catenated)*** strands of DNA formed

\

11. Type II Topoisomerase needed to separate the 2 DNA

Polymerase III Structure

* Multiple Subunits
* 2 Polymerases
* Has SSBP
* Clamp-loading complex

\
* Middle Round Structure
* DnaB Helicase
* DnaG Primase

DNA Ligase Mechanism

1. Lysine attaches to phosphoamide

\

2. Lysine + Phosphoamide attaches to 5’ Phosphate of the nick

\

3. 3’OH neighbouring nucleotide attacks 5’ Phosphate

\

4. AMP is released as a product

\

5. Phosphodiester linkage forms

* Ultimately sealing the nick

\

3’ → 5’ Exonuclease (Acts on Leading Strand)

* E.coli Pol I
* E.coli Pol II
* E.coli Pol III

\
* Allows for proofreading

\
Structure:

* DNA binding site is positively charged

5’ → 3’ Exonuclease (Acts on Lagging Strand)

* ONLY E.coli Pol I

\
* Degrades strand in front of replication complex


* Removes RNA primer in replication and DNA repair

Polymerase III

* Primary enzyme involved in replication
* Most processive

\
* Hydrolysis Reaction
* Water is applied
* Inorganic pyrophosphate PP is released

Polymerase I

* Polymerase critical in:
* Lagging Strand Synthesis
* DNA Repair

Polymerase II

* Polymerase critical in
* DNA repair

DNA Unwinding Elements (DUE)

* Area where replication occurs
* AT rich sections of sequence

DnaA

* Involved in initial unwinding during replication

\
* Is and ATP-binding protein
* Active when ATP is bound

Helicase DnaB

* Binds onto lagging strand
* Bind as ***hexamers***
* NTP-dependent (ATP, CTP GTP)

Sliding Clamp

* Known as Beta-clamp
* Dimer
* Enhances processivity of Polymerase III
* Found on duplex next to polymerase
* Clamps and polymerases have to be loaded each second

\

TerA

* Stops counter-clockwise replication
* Binds to TUS protein

TerC

* Stops clockwise replication
* Binds to TUS protein

Terminus Utilization Substance

* When binded to Ter(A-H) protein, will stop replication in one direction.

Errors in Synthesis

Results from:

* Not having excess of specific nucleotides
* Tight control of base pairing during synthesis
* 3’→5’ exonuclease activity (proof reading)
* Multiple DNA repair mechanisms

Mutations

__**Caused by:**__

* **Radiation Damage**
* Thymine dimers prevent replication (A unable to pair with T)

\
* **Spontaneous Chemical/Oxidative Reaction**

\
* **Transitions (Purine/Purine Substitution)**
* Deamination
* C→U
* 5mC → T

\
* **Transversions (Purine/Pyrimidine)**
* Oxidation
* C8 of Guanine → 8-oxoguanine

\
* **Transition/Transversion**
* Alkylation
* Effect Base-Pairing: O6-methylguanine
* No Effect on Base-Pairing: N6-Methyladenine, 5-Methylcytosine, N4-Methylcytosine

\
* **Insertions/Deletions (Nucleotides Lost/Gained)**
* Loss
* Depurination
* Spontaneous hydrolysis of glycosidic bond creating apurinic site
* Add/Loss
* Intercalating Agents
* Causes spacing issues