Ch6: DNA and Biotech

Deoxyribonucleic Acid (DNA)

Store genetic info

Polydeoxyribonucleotide from linked monodeoxyribonucleotides

In chromosomes, mitochondria, and chloroplasts

Nucleosides

Pentose sugar (C-1’) + nitrogenous base

Nucleotides

Nucleoside (C-5’) + phosphate groups

Named for # of phosphate groups

DNA building blocks

Nucleotides: ADP and ATP

High energy from repulsion of - phosphate groups

Bond breaking = exothermic

Nucleic Acid Classification

Pentose sugar

Ribose: RNA

Deoxyribose: DNA

Sugar-Phosphate Backbone

Read 5’ to 3’

Form from nucleotides joined by phosphodiester bonds 3’-5’ (3’ C to 5’ phosphate)

Overall neg charge

DNA Directionality

Polarity from 5’ and 3’

5’: -OH or phosphate bonded to sugar C-5’

3’: -OH on sugar C-3’

Reading DNA

5’ to 3’: 5’-ATG-3’

3’ to 5’: 3’-GTA-5’

Phosphates: pApTpG

Deoxyribose: dAdTdG

Nitrogenous Base: Purines

**PURE AGriculture**

2 rings

Adenine (A) and guanine (G)

In DNA and RNA

Nitrogenous Base: Pyrimidines

**PYRAMIDS can CUT**

1 ring

Cytosine (C), thymine (T), uracil (U)

In DNA (C and T) and RNA (U)

Aromatic

Stable ring (From delocalized π e)

1. Cyclic compound

2. Planar compound

3. Conjugated (alternate single and double bonds)

4. 2n+2 π e (Huckel’s rule)

Heterocycles

Ring structures with 2+ different elements in ring

Aromatic Heterocycles

Nitrogenous base structure give nucleic acids stability

Watson-Crick DNA Model

Antiparallel double helix

Sugar-phosphate backbone outside, nitrogenous bases inside

Complementary base-pairing

Complementary Base-Pairings

A and T/U: 2 H bonds

G and C: 3 H bonds (stronger)

Chargaff’s Rule

In DNA:

%A = %T

%C = %G

%purines = %pyrimidines

B-DNA

Right-handed DNA helix

Grooves between strands (protein binding sites)

Z-DNA

Left-handed zigzag DNA helix

From high GC-content or high salt concentration

No biological activity

DNA Denaturation

Disrupt H bonds and base-pairings

Intact covalent bonds

Separate single DNA strands from helix

Denaturation Agents

Heat, alkaline pH, chemicals (formaldehyde and urea)

DNA Reannealing

Repairing single-stranded DNA into helix

Remove denaturing conditions

Nucleoproteins

Proteins associating with DNA

Acid soluble and stimulate transcription

Histones

Nucleoprotein (basic)

Wind DNA into chromatin

5 proteins (H1, H2A, H2B, H3, H4)

Nucleosome

Histone core with 2 protein copies (H2A, H2B, H3, H4)

200 DNA base pairs wrapped around

H1 Protein

Seal DNA entering and leaving nucleosome

Increase stability

Chromatin

DNA associated with histones

Heterochromatin

Condensed chromatin around histones (small amount)

Repetitive DNA sequences

Dark under microscope

Transcriptionally silent

Euchromatin

Uncondensed chromatin unbound from histones

Light under microscope

Genetically active

Telomere

Repeating TTAGGG end sequence

Lost during replication to prevent info loss

Prevent unraveling (high GC-content)

Telomerase

Replace lost telomeres

Centromere

Chromosome centre

Site of construction

Contain heterochromatin with high GC-content (connect sister chromatids until separated by microtubules)

Replication: Replisome/Replication Complex

Specialized proteins assisting DNA polymerase

Replication: Origin of Replication

DNA unwinding site

Replication forks form on either side

Origin of Replication: Prokaryotes

1 origin

Produce 2 identical circular DNA molecules

Origin of Replication: Eukaryotes

Multiple origins

Produce sister chromatids connected at centromere

Replication: Helicase

Unwind DNA

Replication: Single-Stranded DNA-Binding Proteins

Bind unraveled DNA to prevent reassociation and degradation by nucleases

Replication: DNA Topoisomerase

Negatively supercoil DNA to relieve positive supercoiling from DNA unwinding

Cut and reseal strands

Semiconservative Replication

1 retained parent strand + 1 new daughter strand makes new DNA molecule

Replication: DNA Polymerase

Read DNA template (parent strand) 3’ to 5’

Synthesize complementary daughter strand 5’ to 3’

Replication: Leading Strand

Continuous replication in replication fork direction

3’ to 5’ parent strand

Replication: Lagging Strand

Broken replication in opposite direction of replication fork

5’ to 3’ parent strand

Synthesize Okazaki fragments

More prone to mutations (more replication starts/stops and more primers)

Replication: Primase

Add RNA primer (5’ to 3’) for DNA polymerase

1 on leading, multiple on lagging

Replication: Prokaryote DNA Polymerase Synthesize Strands

DNA polymerase III

Replication: Eukaryote DNA Polymerase Synthesize Strands

DNA polymerases α, δ, ε

Replication: Incoming Nucleotides

5’ deoxyribonucleotide triphosphate (dATP, dCTP, dGTP, dTTP)

Replication: Bond Formation

Release pyrophosphate (PPi)

Replication: Removing RNA

Prokaryotes: DNA polymerase I

Eukaryotes: RNase H

Replication: Replacing RNA Primer with DNA

Prokaryotes: DNA polymerase I

Eukaryotes: DNA polymerase δ

Replication: DNA Ligase

Seal DNA ends

Replication: DNA Polymerase γ

Eukaryotes

Replicate mitochondrial DNA

Replication: DNA Polymerase β and ε

Eukaryotes

DNA repair

Replication: DNA Polymerase δ and ε

Eukaryotes

PCNA protein assists to form sliding clamp

Strengthen DNA polymerase and template strand interaction

Cancer Cells

From mutated genes

Excessive proliferation

Infect local (metastasis) or distant (bloodstream/lymph) tissues

Oncogenes

Mutated genes causing cancer (1 allele)

Code cell cycle-related proteins

Ex: Src (sarcoma)

Proto-Oncogenes

Pre-mutated oncogenes

Tumor Suppressor Genes (Antioncogenes)

Stop tumour progression

Code proteins inhibiting cell cycle or for DNA repair

Mutation loses tumor suppression activity (2 alleles)

Ex: p53, Rb (retinoblastoma)

Proofreading

In S phase

DNA polymerase detect instability from mismatched H bonds between incorrect paired bases

Excise and replace base

Proofreading: Differentiating Parent and Daughter Strands

Methylation level

Template strand older = more methylated

Mismatch Repair

In G2 phase

Enzymes detect and remove replication errors

Eukaryote Genes: MSH2 and MLH1

Prokaryote Genes: MutS and MutL

Nucleotide Excision Repair (NER) Mechanism

In G1 and G2 phases

Remove lesions distorting DNA helix

Ex: Thymine dimers caused by UV light

NER 1: Scan DNA

Identify bulges in strand

NER 2: Excision Endonuclease

Cut phosphodiester backbone to remove thymine dimer

NER 3: DNA Polymerase and DNA Ligase

Fill gap and seal strand

Base Excision Repair

In G1 and G2 phases

Remove lesions not distorting DNA helix

Ex: Uracil from cytosine deamination caused by thermal energy absorption

Base Excision Repair 1: Glycosylase Enzyme

Recognize and remove uracil

Leave apurinic/apyrimidinic (AP) or abasic site

Base Excision Repair 2: AP Endonuclease

Recognize and remove damaged sequence in AP site

Base Excision Repair 3: DNA Polymerase and DNA Ligase

Fill gap and seal strand

Recombinant DNA

DNA fragments from different sources

Gene cloning and polymerase chain reaction (PCR)

DNA Cloning

Produce large amounts of desired DNA sequence

DNA Cloning 1: Recombinant Vector

Restriction enzyme cleave vector plasmid (bacterial/viral) and DNA

Ligate DNA to vector plasmid

Transfer to host bacterium

DNA Cloning 2: Bacteria Colonies

Multiply DNA in vector

DNA Cloning 3: Isolate Colony

Include antibiotic resistance gene in recombinant vector

Grow many colonies

DNA Cloning 4: Express Gene

Generate large amounts of recombinant protein

DNA Cloning 4: Lyse Bacteria

Isolate and replicate recombinant vector

Restriction enzyme processing release cloned DNA

Restriction Enzymes/Endonucleases

Recognize and cleave at palindromic sequences

Isolated from source bacteria

Restriction Enzymes: Sticky Ends

Offset cuts

Facilitate recombination of restriction fragment with vector DNA

DNA Libraries

Produced from cloning

Known DNA sequence collection

Genomic or cDNA

Genomic Libraries

Large DNA fragments

Coding (exon) and noncoding (intron) regions

CANNOT make recombinant proteins or for gene therapy

cDNA (Complementary/Expression) Libraries

Reverse-transcribing mRNA

Coding (exon) regions only

CAN make recombinant proteins or for gene therapy

Hybridization

Joining complementary base pair sequences

DNA-DNA or DNA-RNA recognition

Hybridization: PCR

Produce DNA copies by hybridization without bacteria

Amplify sequence between primers

PCR Materials

Template DNA

Primers

Deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, dTTP)

DNA polymerase

PCR 1: Denature

Heat and separate double helix

PCR 2: Anneal

Primers complementary to sequences flanking region anneal to DNA

High GC-content for H bond stability

PCR 3: Extension

DNA polymerase withstanding high temps add complementary nucleotides

Cool to reanneal daughter and parent strands

Hybridization: Gel Electrophoresis

Separate macromolecules by size and charge

Agarose Gel Electrophoresis

Negative DNA migrate to anode

Longer strand = slower migration

Hybridization: Southern Blot

Detect DNA presence and quantity

Southern Blot 1: Gel Electrophoresis

Restriction enzymes cut DNA

Separate by gel electrophoesis

Southern Blot 2: Membrane Transfer

Separated DNA transferred from gel to membrane

Souther Blot 3: Probing

ssDNA probes bind to complementary sequences on membrane

Probes labeled with radioisotopes/indicators for detection

DNA Sequencing Materials

Template DNA

Primers

DNA polymerase

Deoxyribonucleotide triphosphates

Dideoxyribonucleotides (ddATP, ddCTP, ddGTP, ddTTP) → H at C-3’

DNA Sequencing 1: DNA Fragments

Produce fragments terminating in dideoxyribonucleotide (H on C-3’ prevent DNA polymerase adding nucleotides)

DNA Sequencing 2: Gel Electrophoresis

Separate fragments by size

DNA Sequencing 3: Reading

Read last base for each fragment in order to identify whole DNA sequence

Biotech Applications: Gene Therapy

Potential cure for inherited diseases

Transfer normal gene copy in viral vector to replace mutated/inactive

Transgenic Animal Models

Alter germ line from transgene (cloned gene) introduction

Knockout Animal Models

Delete gene

Transgene: Fertilized Ovum

Gene injected into nucleus

Implant into surrogate mother to produce offspring with transgene germline

Study dominant gene effects