DNA STRUCTUREDNA STRUCTURE

NUCLEIC ACIDSNUCLEIC ACIDS

Nucleic acids are polymersNucleic acids are polymers Monomer---nucleotidesMonomer---nucleotides

Nitrogenous basesNitrogenous bases PurinesPurines PyrimidinesPyrimidines

Sugar Sugar RiboseRibose DeoxyriboseDeoxyribose

PhosphatesPhosphates +nucleoside=nucleotide+nucleoside=nucleotide

}Nucleosides

The Sugars The Sugars

The Bases The Bases

PURINES

PYRIMIDINES

Bases of DNA (and RNA)Bases of DNA (and RNA)Bases of DNA (and RNA)Bases of DNA (and RNA)

RNA only DNA only

Purines:

Pyrimidines:

Nucleotides and Nucleotides and NucleosidesNucleosides

Chemical Structure of DNA and RNA

Chemical Structure of DNA and RNA

Figure 4.1

RNA DNA

Nucleotide

Nucleoside

1’

2’

4’

The C is named 1’-5’

Resume

Nucleotides and Nucleotides and NucleosidesNucleosides

BASEBASE NUCLEOSIDENUCLEOSIDE DEOXYNUCLEOSIDEOXYNUCLEOSIDEDE

AdenineAdenine AdenosineAdenosine 2-2-deoxyadenosinedeoxyadenosine

GuanineGuanine GuanosineGuanosine 2-2-deoxyguanosinedeoxyguanosine

CytosineCytosine CytodineCytodine 2-deoxycytodine2-deoxycytodine

UracilUracil UridineUridine Not usually found Not usually found

ThymineThymine Not usually foundNot usually found 2-deoxythymidine2-deoxythymidine

Nucleotides are nucleosides + phosphate

NucleotidNucleotide e

Analogs Analogs as Drugsas Drugs

make up 13-34% of the dry weight in bacteriadeoxyribonucleic acid (DNA) and ribonucleic acid (RNA)

O

C C

C C

Base

H

H or OH

H H

H

H

CH2OP

OH

O

HO

Nucleotide: a building block

Sugar:• RNA – ribose (OH)• DNA – deoxyribose (H)

Bases:• adenine (A), cytosine (C), guanine (G), thymine (T)• RNA uses uracil (U) instead of thymine

Nucleoside: base + sugar

• certain nucleotides serve as a storage of energy and reducing powere.g. ATP -> ADP -> AMP

hydrolysis (energy is released)

Nucleic Acids

DNA Stabilization– DNA Stabilization– Complementary Base PairingComplementary Base Pairing

DNA Stabilization-Base DNA Stabilization-Base StackingStacking

DNA Stabilization--H-bonding DNA Stabilization--H-bonding between DNA base pair stacks between DNA base pair stacks

Advantages to Double HelixAdvantages to Double Helix

Stability---protects bases from attack Stability---protects bases from attack by Hby H22O soluble compounds and HO soluble compounds and H22O O itself.itself.

Provides easy mechanism for Provides easy mechanism for replicationreplication

Physical Structure (cont’d)Physical Structure (cont’d)

Chains are anti-parallel (i.e in opposite Chains are anti-parallel (i.e in opposite directions)directions)

Diameter and periodicity are Diameter and periodicity are consistent consistent 2.0 nm2.0 nm 10 bases/ turn10 bases/ turn 3.4 nm/ turn3.4 nm/ turn

Width consistent because of Width consistent because of pyrimidine/purine pairingpyrimidine/purine pairing

Physical Structure (cont’d)Physical Structure (cont’d)

G-C ContentG-C Content

A=T, G=C, but ATA=T, G=C, but AT≠GC≠GC Generally GC~50%, but extremely Generally GC~50%, but extremely

variablevariable EX.EX.

Slime mold~22%Slime mold~22% Mycobacterium~73%Mycobacterium~73%

Distribution of GC is not uniform in Distribution of GC is not uniform in genomesgenomes

CONSEQUENCES OF GC CONSEQUENCES OF GC CONTENTCONTENT

GC slightly denser GC slightly denser Higher GC DNA moves further in a Higher GC DNA moves further in a

gradientgradient Higher # of base pairs=more stable Higher # of base pairs=more stable

DNA, i.e. the strands don’t separate DNA, i.e. the strands don’t separate as easily. as easily.

FORMS OF DNAFORMS OF DNA

Supercoiling Supercoiling

Cruciform Cruciform StructuresStructures

Another adaptation to supercoilingAssociated with palindromes

DNA is DynamicDNA is Dynamic Like proteins, DNA has 3Like proteins, DNA has 3ºº structure structure Why so many deviations from normal Why so many deviations from normal

conformation? conformation? Effects on transcription (gene Effects on transcription (gene

expression)expression) Enhances responsivenessEnhances responsiveness May also serve in packagingMay also serve in packaging

NOTE: most cellular DNA exists as NOTE: most cellular DNA exists as protein containing supercoilsprotein containing supercoils

Denaturation of DNADenaturation of DNA Denaturation by Denaturation by

heating.heating. How observed?How observed?

AA260260 For dsDNA, For dsDNA,

AA260260=1.0 for 50 =1.0 for 50 µg/mlµg/ml For ssDNA and RNA For ssDNA and RNA

AA260260=1.0 for 38 =1.0 for 38 µg/mlµg/ml For ss oligosFor ss oligos

AA260260=1.0 for 33 =1.0 for 33 µg/mlµg/ml Hyperchromic shiftHyperchromic shift

The T at which ½ the DNA sample is denatured is called the melting temperature (Tm)

Importance of TImportance of Tmm

Critical importance in any technique Critical importance in any technique that relies on complementary base that relies on complementary base pairing pairing Designing PCR primersDesigning PCR primers Southern blotsSouthern blots Northern blotsNorthern blots Colony hybridization Colony hybridization

Factors Affecting TFactors Affecting Tmm

G-C content of sampleG-C content of sample Presence of intercalating agents Presence of intercalating agents

(anything that disrupts H-bonds or (anything that disrupts H-bonds or base stacking)base stacking)

Salt concentrationSalt concentration pH pH LengthLength

RenaturationRenaturation

Strands can be induced to renature Strands can be induced to renature (anneal) under proper conditions. (anneal) under proper conditions. Factors to consider:Factors to consider: TemperatureTemperature Salt concentrationSalt concentration DNA concentrationDNA concentration TimeTime

CCoot Curvest Curves

What Do CWhat Do Coot Curves Reveal?t Curves Reveal?

Complexity of DNA sampleComplexity of DNA sample Reveals important info about the Reveals important info about the

physical structure of DNAphysical structure of DNA Can be used to determine TCan be used to determine Tmm for for

techniques that complementary base techniques that complementary base pairing. pairing.

Complexity of DNA- FactorsComplexity of DNA- FactorsRepetitive Sequences Repetitive Sequences

Single Copy GenesSingle Copy Genes Highly repetitive (hundreds to millions)Highly repetitive (hundreds to millions)

Randomly dispersed or in tandem repeatsRandomly dispersed or in tandem repeats Satellite DNASatellite DNA

Microsatellite repeatsMicrosatellite repeats Miniisatellite repeatsMiniisatellite repeats

Middle repetitive (10- hundreds)Middle repetitive (10- hundreds) ClusteredClustered DispersedDispersed

Slightly repetitive (2-10 copies)Slightly repetitive (2-10 copies)

Highly repetitive sequences

Middle repetitiveMiddle repetitive sequences

Unique sequences

Renaturation curves of E. coli and calf DNA

RNA RNA

TypesTypes mRNAmRNA tRNAtRNA rRNArRNA

It’s still an RNA worldIt’s still an RNA world snRNAsnRNA siRNAsiRNA RibozymesRibozymes

Behavior in AcidsBehavior in Acids Dilute or mild acidic conditionsDilute or mild acidic conditions Intermediate conditions. EX. 1N HCl Intermediate conditions. EX. 1N HCl

@ 100@ 100ºC for 15m : DepurinationºC for 15m : Depurination Harsher treatment-EX. 2-6N HCl, Harsher treatment-EX. 2-6N HCl,

higher temps: Depyrimidination.higher temps: Depyrimidination. NOTE: some phosphodiester bond NOTE: some phosphodiester bond

cleavage observed during cleavage observed during depurination, much more during depurination, much more during depyrimidinationdepyrimidination

Behavior in BasesBehavior in Bases N-glycosidic bonds stable in mild N-glycosidic bonds stable in mild

alkaline conditionsalkaline conditions DNA meltsDNA melts PhosphodiesterPhosphodiester linkages in DNA and linkages in DNA and

RNA show very different behavior in RNA show very different behavior in weak bases (EX 0.3 N KOH @37weak bases (EX 0.3 N KOH @37ººC ~1 C ~1 hr.)hr.)

RNA Hydrolysis in Alkaline RNA Hydrolysis in Alkaline SolutionsSolutions

2,3 cyclic nucleotide

Hydrolysis by EnzymesHydrolysis by Enzymes

Nuclease—catalyzes hydrolysis of Nuclease—catalyzes hydrolysis of phosphodiester backbone phosphodiester backbone ExonucleasesExonucleases EndonucleasesEndonucleases

General. Ex DNAse IGeneral. Ex DNAse ISpecific Ex. Restriction Specific Ex. Restriction endonucleasesendonucleases

RibozymesRibozymes

Restriction Restriction EnzymesEnzymes

RIBOZYMERIBOZYMESS

Catalytic Catalytic RNARNA

Can work Can work alone or with alone or with proteinsproteins

Therapeutic Therapeutic applications?applications?

SEQUENCINGSEQUENCING

Purpose—determine nucleotide Purpose—determine nucleotide sequence of DNA sequence of DNA

Two main methodsTwo main methods Maxam & Gilbert, using chemical

sequencing Sanger, using dideoxynucleotides

The Sanger TechniqueThe Sanger Technique Uses Uses

dideoxynucleotidedideoxynucleotides (dideoxyadenine, s (dideoxyadenine, dideoxyguanine, dideoxyguanine, etc)etc)

These are These are molecules that molecules that resemble normal resemble normal nucleotides but nucleotides but lack the normal -lack the normal -OH groupOH group..

Because they lack the -OH (which Because they lack the -OH (which allows nucleotides to join a allows nucleotides to join a growing DNA strand), replication growing DNA strand), replication stops.stops.

Normally, this wouldbe where another phosphateIs attached, but with no -OHgroup, a bond can not form and replication

stops

The Sanger Method RequiresThe Sanger Method Requires Multiple copies of single stranded Multiple copies of single stranded

template DNAtemplate DNA A suitable A suitable primerprimer (a small piece of DNA (a small piece of DNA

that can pair with the template DNA to that can pair with the template DNA to act as a starting point for replication)act as a starting point for replication)

DNA polymeraseDNA polymerase (an enzyme that copies (an enzyme that copies DNA, adding new nucleotides to the 3’ DNA, adding new nucleotides to the 3’ end of the templateend of the template

A ‘pool’ of A ‘pool’ of normal nucleotidesnormal nucleotides A small proportion of A small proportion of dideoxynucleotidesdideoxynucleotides

labeled in some way ( radioactively or labeled in some way ( radioactively or with fluorescent dyes) with fluorescent dyes)

The template DNA pieces are replicated, The template DNA pieces are replicated, incorporating normal nucleotides, but incorporating normal nucleotides, but occasionally and at randomoccasionally and at random dideoxy dideoxy (DD) nucleotides are taken up.(DD) nucleotides are taken up.

This stops replication on that piece of This stops replication on that piece of DNADNA

The result is a The result is a mix of DNA lengthsmix of DNA lengths, each , each ending with a particular labeled ending with a particular labeled DDnucleotide.DDnucleotide.

Because the different lengths ‘travel’ at Because the different lengths ‘travel’ at different rates during electrophoresis, different rates during electrophoresis, their order can be determined.their order can be determined.

Termination during Replication

DNA SEQUENCE 3’

G C A T T G G G A A C C

PRIMER 5’

C G T A

NO OF BASES

1 2 3 4 5 6 7 8 9 10 11 12

G terminated C G T A A C C T T G

C G T A A C C T T G G A terminated

C G T A A Tterminated C G T A A C C T

C G T A A C C T T

C terminated C G T A A C C G T A A C C C G T A A C C C