8/13/2016 Chapter 16 DNA molecule responsible for all cell - - PDF document

8/13/2016 1 Chapter 16 DNA The Molecular Basis

• f

Inheritance

DNA molecule responsible for all cell activities and contains the genetic code Genetic ic Code method cells use to store the program that is passed from one generation to another

DISCOVERY OF THE GENETIC CODE

1928: Frederick Griffith

Transformation

1928: Frederick Griffith

• 1. Grew 2 strains of bacteria on

plates

• smooth colonies- caused

disease (virule lent)

• rough edge colonies-did not

cause disease (aviru rule lent)

• 2. Injected into mice

Results:

• smooth colonies: died
• rought colonies: lived

Conclusion: bacteria didn’t produce a toxin to kill mice

Experiment 1

Experiment 2

• 1. Injected mice with heat

killed virulent strain 2. Injected mice with non -virulent strain + heat killed virulent strain Results:

• heat killed: lived
• mixed strains: mice

developed pneumonia Conclusion: heat killed virulent strain passed disease causing abilities to non virulent strain

After Experiment

Cultured bacteria from dead mice and they grew virulent strain. Griffith hypothesized that a factor was transferred from heat killed cells to live cells .

TRANS NSFOR FORMA MATIO ION

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1944: Avery (et al)

• 1. Repeated Griffith’s experiment with same results.
• result: transformation occurred
• 2. Did a second experiment using enzymes that would

destroy RNA.

• result: transformation occurred
• 3. Did third experiment using enzymes that would destroy DNA.
• result: no transformation

CONCLUSION DNA was transforming factor

1952: Hershey / Chase

• studied how viruses (bacteriophage) affect bacteria.

Bacterio

riophage ge Virus composed of DNA core and protein coat that infect bacteria

Hershey Chase Experiment

• 1. They labeled virus protein

coat with radioactive sulfur

• 2. They labeled virus DNA

with radioactive phosphorous Result lt

• bserved that bacteria had

phosphorous *** virus injected bacterial cells with its phosphorous labeled DNA*** Conclu clusion DNA carried genetic code since bacteria made new

• DNA. animation

DISCOV OVERY RY OF STRUCTU TURE OF DNA

Early 1950’s: Rosalind Franklin (English)

x ray cryst stallogra graphy hy eviden dence ce: X pattern showed that fibers of DNA twisted and molecules are spaced at regular intevals on length fiber.

Maurice Wilkins: x ray diffraction, worked with Franklin

Same time period:

Chargaff (American biochemist)

Chargaff’s Rule:

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1953 Watson (American) & Crick (English)

**double helix model** won Nobel prize in 1962 Discovered the double helix by building models to conform to Franklin’s X-ray data and Chargaff’s Rules

DNA

• double strand of nucleotides
• may have 1000’s of nucleotides in 1 strand

(very long molecule)

• bases join up in specific (complementary) pairs:
• complementary pairs (base pairing rules)

1 purine bonds with 1 pyrimid idin ine on one rung of the ladder connected by a weak H bond

C - G A – T

Order r of nucleotid ides not import rtant, proper r comple lementary ry bases must be paired.

STRUCT CTURE URE OF DNA

Backbone Phosphate + Deoxyribose sugar (5 C) Rungs 4 Nitrogenous bases

• Purines

Adenine A Guanine G

• Pyrimidines

Thymine T Cytosine C D bases attached to sugar

• E. bases attached to each
• ther by weak H bond

Nucleotide Structure

Purines

Pyrimidines Sugar Base Phosphate

DNA makes up Chromosomes

(chromatin packing)

DNA Comparison

Prokaryotic DNA

• Double-stranded
• Circular
• One chromosome
• In cytoplasm
• No histones
• Supercoiled DNA

Eukaryotic DNA

• Double-stranded
• Linear
• Usually 1+ chromosomes
• In nucleus
• DNA wrapped around

histones (proteins)

• Forms chromatin

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REPLIC ICATION ION OF DNA

Process of duplication of DNA

• Before cell can divide a new copy of DNA

must be made for the new cell

• Semiconser

ervative ive replication ion: each strand acts as a template (pattern) for new strand to be made End Result lt:

• ne old strand, one new daughter strand

DNA REPLICATION

Models of DNA Replication

Discovery of Replication Model

Meselson and Stahl

• Cultured E coli with heavy

isotope 15N for many generations.

• Transferred to light isotope

14N

• Sampled after first and

second replication

• Removed bacteria and extracted DNA
• Centrifuged to separate different density DNA

Meselson and Stahl

• Compared results to

models of replication

• 1st replication:

Hybrid DNA (14 and 15)

• Eliminated

conservative model

• 2nd replication:

Light and hybrid DNA

• Eliminated dispersive

model

• Supported semi-conservative model of replicaiton.

Steps of Replication

1. Enzyme DNA helica case attaches to DNA molecule at origins of replication and breaks H bonds so strand unwinds

• single strand binding proteins bind to unpaired bases to keep

them from re-binding

• topoisomerase relieves additional strain on forward DNA by

breaking and rejoining DNA strands

• replica

icatio ion forks: two areas on either end of the DNA where double helix separates

• forms replica

catio ion bubble: “bubble” under electron microscope

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• 2. enzyme RNA Primase

lays down an RNA primer on DNA strand

• RNA primer segment

signals beginning of replication

• 3. Enzyme DNA polymera

rase III moves along each of DNA strand and adds complementary bases of nucleotides floating freely in nucleus

• A. DNA polymerase III begins

synthesis at RNA primer segment

• B. DNA polymerase I replaces

RNA primers with DNA nucleotides

DNA Directionality

antiparallel strands

5’ 3’ 5’ 3’

• Directionality: DNA

polymerase III reads the template in the 3’ to 5’ direction Daughter DNA strand (since it is complementary) must be synthesized in the 5’ to 3’ direction Strands are antiparallel. But if there exist no DNA polymerases capable of polymerizing DNA in the 3' to 5' direction, how could this be? Discontinuous synthesis

• synthesis only occurs when a

large amount of single strand DNA is present

• daughter DNA is then synthesized

in 5’ to 3’ direction

• leading and lagging strands:
• leading strand – continuously

synthesized DNA strand

• lagging strand - delayed,

fragmented, daughter DNA

• Okazaki fragments-

discontinuous fragmented DNA segments

• D. DNA ligase stitches

together Okazaki fragments into a single, unfragmented daughter molecule

• E. enzyme chops off

RNA primer and replaces it with DNA

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• 3. DNA polymerase III catalyzes

formation of H bonds between nucleotides of template and newly arriving nucleotides which will form daughter DNA 4. Once all DNA is copied, daughter DNA detaches

Animation DNA Replication Fork Okazaki fragment animation

End Replication Problem

• On one end, RNA primer

cannot be replaced with DNA because it is a 5’ (DNA polymerase III can only read from 3’ to 5’)

• Causes daughter DNA’s

to be shorter with each replication (cell division)

Solution to End Replication Problem

telomere res: repeated units of non-coding short nucleotide sequences (TTAGGG) at ends of DNA

• become shorter with repeated cell divisions
• once telomeres are gone, coding sections of chrom.

are lost and cell does not have enough DNA to function ***telomere theory of aging***

• telomera

rase: special enzyme that contains an RNA template molecule so that telomeres can be added back on to DNA (rebuilds telomeres) ** found in: Cancer cells - immortal in culture Stem cells ** not found in most differentiated cells

Telomeres and Telomerase Speed of Replication

• Multiple replication forks- replication occurs simultaneously on many

points of the DNA molecule

• Would take 16 days to replicate 1 strand from one end to the other on

a fruit fly DNA without multiple forks

• Actually takes ~ 3 minutes / 6000 sites replicate at one time
• Human chromosome replicated in about 8 hours with multiple

replication forks working together

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Accuracy and Repair

• DNA polymerases proofread as bases are added
• can remove damaged nucleotides and replace

with new ones for accurate replication

• Mismatch repair: special enzymes fix incorrect

pairings

• Nucleotide excision repair:
• Nucl

clea eases ses cut damaged DNA

• DNA polymerase and ligase fill in gaps

RNA does not have this ability- reason RNA viruses mutate so much

Nucleotide Excision Repair

Errors: Pairing errors: 1 in 100,000 nucleotides Complete DNA: 1 in 10 billion nucleotides

Importance of DNA

1. Controls formation of all substances in the cell by the genetic code 2. Directs the synthesis of specific strands of m RNA to make proteins

RNA A (Ribonucleic acid)

Another nucleic acid takes orders from DNA Used in protein synthesis