DNA: The Genetic MaterialDNA: The Genetic MaterialChapter 14

Griffith’s experiment with Streptococcus pneumoniae◦Live S strain cells killed the mice◦Live R strain cells did not kill the mice◦Heat-killed S strain cells did not kill the

mice◦Heat-killed S strain + live R strain cells

killed the mice

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Transformation◦Information specifying virulence passed

from the dead S strain cells into the live R strain cells

Our modern interpretation is that genetic material was actually transferred between the cells

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Avery, MacLeod, & McCarty Avery, MacLeod, & McCarty – 1944– 1944

Repeated Griffith’s experiment using purified cell extracts

Removal of all protein from the transforming material did not destroy its ability to transform R strain cells

DNA-digesting enzymes destroyed all transforming ability

Supported DNA as the genetic material

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Hershey & Chase Hershey & Chase ––1952 1952 Investigated bacteriophages

◦Viruses that infect bacteriaBacteriophage was composed of only

DNA and proteinWanted to determine which of these

molecules is the genetic material that is injected into the bacteria

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Bacteriophage DNA was labeled with radioactive phosphorus (32P)

Bacteriophage protein was labeled with radioactive sulfur (35S)

Radioactive molecules were tracked Only the bacteriophage DNA (as indicated by

the 32P) entered the bacteria and was used to produce more bacteriophage

Conclusion: DNA is the genetic material

ChargaffChargaff’’s Ruless Rules

Erwin Chargaff determined that◦Amount of adenine = amount of thymine◦Amount of cytosine = amount of guanine◦Always an equal proportion of purines (A

and G) and pyrimidines (C and T)

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Rosalind FranklinRosalind Franklin

Performed X-ray diffraction studies to identify the 3-D structure◦Discovered that DNA is

helical◦Using Maurice Wilkins’ DNA

fibers, discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm

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James Watson and Francis Crick – James Watson and Francis Crick – 1953 1953

Deduced the structure of DNA using evidence from Chargaff, Franklin, and others

Did not perform a single experiment themselves related to DNA

Proposed a double helix structure

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DNA StructureDNA Structure DNA is a nucleic acid Composed of nucleotides

◦ 5-carbon sugar called deoxyribose◦ Phosphate group (PO4)

Attached to 5′ carbon of sugar◦ Nitrogenous base

Adenine, thymine, cytosine, guanine◦ Free hydroxyl group (—OH)

Attached at the 3′ carbon of sugar

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Pu

rin

esP

yrim

idin

esAdenine Guanine

NH2CC

NN

N

C

HN

CCH

O

H

H

OC

NC

HN

CNH2

H

CH O

O

C

NC

HN

CH3C

CHH

O

O

C

NC

HN

CH

CH

NH2

CC

NN

N

C

HN

CCH

H

Nitrogenous Base

4´

5´

1´

3´ 2´

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7 6

394

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O

P

O–

–O

Phosphate group

Sugar

Nitrogenous base

O CH2

N N

O

NNH2

OH in RNA

Cytosine(both DNA and RNA)

Thymine(DNA only)

Uracil(RNA only)

OHH in DNA

Phosphodiester bond◦ Bond between adjacent

nucleotides◦ Formed between the

phosphate group on the 5’ carbon of one nucleotide and the 3′ —OH of the next nucleotide

The chain of nucleotides has a 5′-to-3′ orientation

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BaseCH2

O

5´

3´

O

P

O

OH

CH2

–O O

C

Base

O

PO4

Phosphodiesterbond

Double helixDouble helix2 strands are polymers of

nucleotidesPhosphodiester backbone –

repeating sugar and phosphate units joined by phosphodiester bonds

Wrap around 1 axisAntiparallel

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Complementarity of bases

A forms 2 hydrogen bonds with T

G forms 3 hydrogen bonds with C

Gives consistent diameter

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A

H

Sugar

Sugar

Sugar

Sugar

T

G C

N

H

N O

H

CH3

H

HN

N N H N

N

N

H

H

H

N O H

H

H N

N H

N

N HN N

Hydrogenbond

Hydrogenbond

Conservative Semiconservative Dispersive

DNA ReplicationDNA Replication3 possible models

1. Conservative model2. Semiconservative model3. Dispersive model

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Meselson and Stahl – 1958 Meselson and Stahl – 1958 Bacterial cells were grown in a

heavy isotope of nitrogen, 15NAll the DNA incorporated 15N Cells were switched to media

containing lighter 14NDNA was extracted from the cells at

various time intervalsThe semiconservative method was

confirmed

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DNA ReplicationDNA ReplicationRequires 3 things

◦Something to copy Parental DNA molecule

◦Something to do the copying Enzymes

◦Building blocks to make copy Nucleotide triphosphates

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DNA replication includes◦Initiation – replication begins◦Elongation – new strands of DNA are

synthesized by DNA polymerase◦Termination – replication is terminated

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DNA polymerase◦Matches existing DNA bases with

complementary nucleotides and links them◦All have several common features Add new bases to 3′ end of existing

strands Synthesize in 5′-to-3′ direction Require a primer of RNA

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5´

3´

5´

5´ 5´3´

3´

RNA polymerase makes primer DNA polymerase extends primer

Prokaryotic ReplicationProkaryotic ReplicationE. coli modelSingle circular molecule of DNAReplication begins at one origin of

replicationProceeds in both directions around the

chromosomeReplicon – DNA controlled by an origin

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E. coli has 3 DNA polymerases◦DNA polymerase I (pol I) Acts on lagging strand to remove

primers and replace them with DNA◦DNA polymerase II (pol II) Involved in DNA repair processes

◦DNA polymerase III (pol III) Main replication enzyme

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SemidiscontinousSemidiscontinousHelicase opens the double helixDNA polymerase can synthesize only in 1

directionLeading strand synthesized continuously from

an initial primerLagging strand synthesized discontinuously

with multiple priming events◦ Okazaki fragments

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Partial opening of helix forms replication fork

DNA primase – RNA polymerase that makes RNA primer◦RNA will be removed and replaced

with DNA

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Leading-strand synthesis◦Single priming event◦Strand extended by DNA pol III

Processivity – subunit forms “sliding clamp” to keep it attached

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Lagging-strand synthesis◦Discontinuous synthesis

DNA pol III◦RNA primer made by primase for each Okazaki

fragment◦All RNA primers removed and replaced by DNA

DNA pol I◦Backbone sealed

DNA ligaseTermination occurs at specific site

◦DNA gyrase unlinks 2 copies

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5´

3´

Primase

RNA primer

Okazaki fragmentmade by DNApolymerase III

Leading strand(continuous)

DNA polymerase I

Lagging strand(discontinuous)

DNA ligase

Replisome Replisome Enzymes involved in DNA

replication form a macromolecular assembly

2 main components◦Primosome

Primase, helicase, accessory proteins

◦Complex of 2 DNA pol III One for each strand

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Eukaryotic ReplicationEukaryotic ReplicationComplicated by

◦Larger amount of DNA in multiple chromosomes

◦ Linear structureBasic enzymology is similar

◦Requires new enzymatic activity for dealing with ends only

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Multiple replicons – multiple origins of replications for each chromosome◦Not sequence specific; can be adjusted

Initiation phase of replication requires more factors to assemble both helicase and primase complexes onto the template, then load the polymerase with its sliding clamp unit◦ Primase includes both DNA and RNA polymerase◦ Main replication polymerase is a complex of DNA

polymerase epsilon (pol ε) and DNA polymerase delta (pol δ)

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Telomeres Telomeres Specialized structures found on the ends of

eukaryotic chromosomesProtect ends of chromosomes from nucleases

and maintain the integrity of linear chromosomes

Gradual shortening of chromosomes with each round of cell division◦ Unable to replicate last section of lagging strand

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Telomeres composed of short repeated sequences of DNA

Telomerase – enzyme makes telomere of lagging strand using and internal RNA template (not the DNA itself)◦ Leading strand can be

replicated to the endTelomerase

developmentally regulated◦ Relationship between

senescence and telomere length

Cancer cells generally show activation of telomerase

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DNA RepairDNA RepairErrors due to replication

◦DNA polymerases have proofreading ability

Mutagens – any agent that increases the number of mutations above background level◦Radiation and chemicals

Importance of DNA repair is indicated by the multiplicity of repair systems that have been discovered

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DNA RepairDNA RepairFalls into 2 general categories1.Specific repair

◦Targets a single kind of lesion in DNA and repairs only that damage

2.Nonspecific◦Use a single mechanism to repair

multiple kinds of lesions in DNA

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PhotorepairPhotorepair

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Specific repair mechanismFor one particular form of

damage caused by UV lightThymine dimers

◦Covalent link of adjacent thymine bases in DNA

Photolyase◦Absorbs light in visible range◦Uses this energy to cleave

thymine dimer

Excision repairExcision repairNonspecific repairDamaged region is

removed and replaced by DNA synthesis

3 steps1. Recognition of damage2. Removal of the

damaged region3. Resynthesis using the

information on the undamaged strand as a template

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