DNA Structure and Chemistry

a). Evidence that DNA is the genetic informationi). DNA transformation – know this termii). Transgenic experiments – know this processiii). Mutation alters phenotype – be able to define

genotype and phenotypeb). Structure of DNA

i). Structure of the bases, nucleosides, and nucleotidesii). Structure of the DNA double helixiii). Complementarity of the DNA strands

c). Chemistry of DNAi). Forces contributing to the stability of the double helixii). Denaturation of DNA

THE FLOW OF GENETIC INFORMATION

DNA RNA PROTEIN

DNA

1

2 3

1. REPLICATION (DNA SYNTHESIS)2. TRANSCRIPTION (RNA SYNTHESIS)3. TRANSLATION (PROTEIN SYNTHESIS)

Thymine (T)

Guanine (G) Cytosine (C)

Adenine (A)

Structures of the bases

Purines Pyrimidines

5-Methylcytosine (5mC)

[structure of deoxyadenosine]

Nucleoside

Nucleotide

Nomenclature

Purinesadenine adenosineguanine guanosinehypoxanthine inosine

Pyrimidinesthymine thymidinecytosine cytidine

+ribose uracil uridine

Nucleoside NucleotideBase +deoxyribose +phosphate

• polynucleotide chain• 3’,5’-phosphodiester bond

ii). Structure of the DNA double helix

Structure of the DNApolynucleotide chain

5’

3’

A-T base pair

G-C base pair

Chargaff’s rule: The content of A equals the content of T, and the content of G equals the content of C in double-stranded DNA from any species

Hydrogen bonding of the bases

Double-stranded DNA

Major groove

Minor groove

5’ 3’

5’ 3’3’ 5’

“B” DNA

Chemistry of DNA

Forces affecting the stability of the DNA double helix

• hydrophobic interactions - stabilize - hydrophobic inside and hydrophilic outside

• stacking interactions - stabilize - relatively weak but additive van der Waals forces

• hydrogen bonding - stabilize - relatively weak but additive and facilitates stacking

• electrostatic interactions - destabilize - contributed primarily by the (negative) phosphates - affect intrastrand and interstrand interactions - repulsion can be neutralized with positive charges

(e.g., positively charged Na+ ions or proteins)

Stacking interactions

Charge repulsion

Ch

arg

e re

pu

lsio

n

Model of double-stranded DNA showing three base pairs

Denaturation of DNA

Double-stranded DNA

A-T rich regions denature first

Cooperative unwinding of the DNA strands

Extremes in pH or high temperature

Strand separationand formation ofsingle-strandedrandom coils

Electron micrograph of partially melted DNA

• A-T rich regions melt first, followed by G-C rich regions

Double-stranded, G-C rich DNA has not yet melted

A-T rich region of DNAhas melted into asingle-stranded bubble

Hyperchromicity

The absorbance at 260 nm of a DNA solution increases when the double helix is melted into single strands.

260

Ab

sorb

ance

Absorbance maximumfor single-stranded DNA

Absorbancemaximum fordouble-stranded DNA

220 300

100

50

0

7050 90

Temperature oC

Pe

rce

nt

hyp

erc

hro

mic

ity

DNA melting curve

• Tm is the temperature at the midpoint of the transition

Average base composition (G-C content) can bedetermined from the melting temperature of DNA

50

7060 80

Temperature oC

Tm is dependent on the G-C content of the DNA

Pe

rce

nt

hyp

erc

hro

mic

ity

E. coli DNA is 50% G-C

Genomic DNA, Genes, Chromatin

a). Complexity of chromosomal DNAi). DNA reassociationii). Repetitive DNA and Alu sequencesiii). Genome size and complexity of genomic DNA

b). Gene structurei). Introns and exonsii). Properties of the human genome iii). Mutations caused by Alu sequences

c). Chromosome structure - packaging of genomic DNAi). Nucleosomes

ii). Histonesiii). Nucleofilament structureiv). Telomeres, aging, and cancer

DNA reassociation (renaturation)

Double-stranded DNA

Denatured,single-strandedDNA

Slower, rate-limiting,second-order process offinding complementarysequences to nucleatebase-pairing

k2

Faster,zipperingreaction toform longmoleculesof double-strandedDNA

Cot1/2

DNA reassociation kinetics for human genomic DNA

Cot1/2 = 1 / k2 k2 = second-order rate constant Co = DNA concentration (initial) t1/2 = time for half reaction of each

component or fraction

50

100

0

% D

NA

re

ass

oc

iate

d

I I I I I I I I I

log Cot

fast (repeated)

intermediate (repeated)

slow (single-copy)

Kinetic fractions: fast intermediate slow

Cot1/2

Cot1/2

high k2

106 copies per genome ofa “low complexity” sequence

of e.g. 300 base pairs

1 copy per genome ofa “high complexity” sequence

of e.g. 300 x 106 base pairs

low k2

Type of DNA % of Genome Features

Single-copy (unique) ~75% Includes most genes 1

Repetitive Interspersed ~15% Interspersed throughout genome between

and within genes; includes Alu sequences 2

and VNTRs or mini (micro) satellites Satellite (tandem) ~10% Highly repeated, low complexity sequences

usually located in centromeres and telomeres

2 Alu sequences are about 300 bp in length and are repeated about 300,000 times in the genome. They can be found adjacent to or within genes in introns or nontranslated regions.

1 Some genes are repeated a few times to thousands-fold and thus would be in the repetitive DNA fraction

50

100

0

I I I I I I I I I

fast ~10%

intermediate ~15%

slow (single-copy) ~75%

Classes of repetitive DNA

Interspersed (dispersed) repeats (e.g., Alu sequences)

TTAGGGTTAGGGTTAGGGTTAGGG

Tandem repeats (e.g., microsatellites)

GCTGAGG GCTGAGGGCTGAGG

viruses

plasmids

bacteria

fungi

plants

algae

insects

mollusks

reptiles

birds

mammals

Genome sizes in nucleotide pairs (base-pairs)

104 108105 106 107 10111010109

The size of the humangenome is ~ 3 X 109 bp;almost all of its complexityis in single-copy DNA.

The human genome is thoughtto contain ~30,000 to 40,000 genes.

bony fish

amphibians

5’ 3’

promoter region

exons (filled and unfilled boxed regions)

introns (between exons)

transcribed region

translated region

mRNA structure

+1

Gene structure

The (exon-intron-exon)n structure of various genes

-globin

HGPRT(HPRT)

total = 1,660 bp; exons = 990 bp

histone

factor VIII

total = 400 bp; exon = 400 bp

total = 42,830 bp; exons = 1263 bp

total = ~186,000 bp; exons = ~9,000 bp

Properties of the human genome

Nuclear genome

• the haploid human genome has ~3 X 109 bp of DNA• single-copy DNA comprises ~75% of the human genome• the human genome contains ~30,000 to 40,000 genes• most genes are single-copy in the haploid genome• genes are composed of from 1 to >75 exons• genes vary in length from <100 to >2,300,000 bp• Alu sequences are present throughout the genome

Mitochondrial genome

• circular genome of ~17,000 bp• contains <40 genes

Familial hypercholesterolemia• autosomal dominant• LDL receptor deficiency

Alu sequences can be “mutagenic”

From Nussbaum, R.L. et al. "Thompson & Thompson Genetics in Medicine," 6th edition (Revised Reprint), Saunders, 2004.

LDL receptor gene

Alu repeats present within introns

Alu repeats in exons

4

4

4

5

5

5 6

6

6

Alu Alu

AluAlu

X

4 6Alu

unequalcrossing over

one product has a deleted exon 5(the other product is not shown)

Chromatin structure

EM of chromatin shows presence ofnucleosomes as “beads on a string”

Nucleosome structure

Nucleosome core (left)• 146 bp DNA; 1 3/4 turns of DNA• DNA is negatively supercoiled• two each: H2A, H2B, H3, H4 (histone octomer)

Nucleosome (right)• ~200 bp DNA; 2 turns of DNA plus spacer• also includes H1 histone

Histones (H1, H2A, H2B, H3, H4)• small proteins• arginine or lysine rich: positively charged• interact with negatively charged DNA• can be extensively modified - modifications in

general make them less positively chargedPhosphorylationPoly(ADP) ribosylationMethylationAcetylation

Hypoacetylation by histone deacetylase (facilitated by Rb)

“tight” nucleosomes assoc with transcriptional repression

Hyperacetylation by histone acetylase (facilitated by TFs)“loose” nucleosomes assoc with transcriptional activation

Nucleofilament structure

Condensation and decondensation of a chromosome in the cell cycle

Telomeres and aging

Metaphase chromosome

centromere

telomere telomere

telomere structure

young

senescent

Telomeres are protective“caps” on chromosomeends consisting of short5-8 bp tandemly repeatedGC-rich DNA sequences,that prevent chromosomesfrom fusing and causingkaryotypic rearrangements.

(TTAGGG)many

(TTAGGG)few

• telomerase (an enzyme) is required to maintain telomere length in germline cells

• most differentiated somatic cells have decreased levels of telomerase and therefore their chromosomes shorten with each cell division

<1 to >12 kb

Class Assignment (for discussion on Sept 9th)

Botchkina GI, et al.“Noninvasive detection of prostate cancer by quantitative analysis of telomerase activity.”Clin Cancer Res. May 1;11(9):3243-3249, 2005

PDF of article is accessible on the website