dna: the genetic material chapter 14. griffith’s experiment with streptococcus pneumoniae ◦ live...
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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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