Dna Structure And Chemistry 392g

DNA Structure and Chemistry a). Evidence that DNA is the genetic information i). DNA transformation – know this term ii). Transgenic experiments – know this process iii). Mutation alters phenotype – be able to define genotype and phenotype b). Structure of DNA i). Structure of the bases, nucleosides, and nucleotides ii). Structure of the DNA double helix iii). Complementarity of the DNA strands c). Chemistry of DNA i). Forces contributing to the stability of the double helix ii). Denaturation of DNA

THE FLOW OF GENETIC INFORMATION

DNA

2

RNA

3

PROTEIN

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

Structures of the bases Purines

Pyrimidines

Adenine (A)

Thymine (T)

5-Methylcytosine (5mC)

Guanine (G)

Cytosine (C)

Nucleoside

[structure of deoxyadenosine]

Nucleotide

Nomenclature

Base

Nucleoside +deoxyribose

Purines adenine guanine

adenosine guanosine

hypoxanthine

inosine

Pyrimidines thymine cytosine

thymidine cytidine +ribose

uracil

Nucleotide +phosphate

uridine

ii). Structure the Structure of theofDNA DNA doublechain helix polynucleotide

5’

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

A-T base pair Hydrogen bonding of the bases

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

Double-stranded DNA 5’

3’

Major groove Minor groove

“B” DNA 3’

5’

3’

5’

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)

Charge repulsion

Stacking interactions

Charge repulsion

Model of double-stranded DNA showing three base pairs

Denaturation of DNA Double-stranded DNA

Strand separation and formation of single-stranded random coils

Extremes in pH or A-T rich regions high temperature denature first

Cooperative unwinding of the DNA strands

Electron micrograph of partially melted DNA

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

A-T rich region of DNA has melted into a single-stranded bubble

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

Hyperchromicity

Absorbance

Absorbance maximum for single-stranded DNA Absorbance maximum for double-stranded DNA

220

260

300

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

Percent hyperchromicity

DNA melting curve 100

50

0 50

70

90

Temperature oC

• Tm is the temperature at the midpoint of the transition

Percent hyperchromicity

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

E. coli DNA is 50% G-C

50

60

70

80

Temperature oC

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

Genomic DNA, Genes, Chromatin a). Complexity of chromosomal DNA i). DNA reassociation ii). Repetitive DNA and Alu sequences iii). Genome size and complexity of genomic DNA b). Gene structure i). Introns and exons ii). Properties of the human genome iii). Mutations caused by Alu sequences c). Chromosome structure - packaging of genomic DNA i). Nucleosomes ii). Histones iii). Nucleofilament structure iv). Telomeres, aging, and cancer

DNA reassociation (renaturation) Double-stranded DNA

Denatured, single-stranded DNA

k2 Slower, rate-limiting, second-order process of finding complementary sequences to nucleate base-pairing

Faster, zippering reaction to form long molecules of doublestranded DNA

DNA reassociation kinetics for human genomic DNA

% DNA reassociated

Cot1/2 = 1 / k2

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

0

50

Kinetic fractions:

fast (repeated) intermediate (repeated)

Cot1/2 Cot1/2

slow (single-copy) Cot1/2

100

I

I

I

I I I log Cot

fast intermediate slow

I

I

I

106 copies per genome of a “low complexity” sequence of e.g. 300 base pairs

high k2

1 copy per genome of a “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 Highly repeated, low complexity sequences usually located in centromeres and telomeres

Satellite (tandem) 0

~10%

fast ~10% 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. 2

intermediate ~15% 50 slow (single-copy) ~75% 100 1

I

I

I

I

I

I

I

I

I

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

Classes of repetitive DNA

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

GCTGAGG

GCTGAGG

Tandem repeats (e.g., microsatellites) TTAGGGTTAGGGTTAGGGTTAGGG

Genome sizes in nucleotide pairs (base-pairs) plasmids viruses bacteria fungi plants algae insects mollusks bony fish

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

amphibians reptiles

The human genome is thought to contain ~30,000 to 40,000 genes. 104

105

106

107

birds mammals 108

109

1010

1011

Gene structure promoter region

exons (filled and unfilled boxed regions)

+1 introns (between exons) transcribed region mRNA structure 5’

3’ translated region

The (exon-intron-exon)n structure of various genes histone total = 400 bp; exon = 400 bp β -globin total = 1,660 bp; exons = 990 bp HGPRT (HPRT)

total = 42,830 bp; exons = 1263 bp

factor VIII 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

Alu sequences can be “mutagenic” Familial hypercholesterolemia • autosomal dominant • LDL receptor deficiency

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

4

5 6 Alu repeats in exons

unequal crossing over

4

Alu

5

Alu

6

X 4

Alu

5

Alu

6 one product has a deleted exon 5

4

Alu

(the other product is not shown)

6

Chromatin structure

EM of chromatin shows presence of nucleosomes 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 charged Phosphorylation Poly(ADP) ribosylation Methylation Acetylation 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 are protective “caps” on chromosome ends consisting of short 5-8 bp tandemly repeated GC-rich DNA sequences, that prevent chromosomes from fusing and causing karyotypic rearrangements.

Telomeres and aging Metaphase chromosome

telomere

centromer e

telomere structure

telomere <1 to >12 kb (TTAGGG)many

(TTAGGG)few

young

senescent

• 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

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