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