Download DNA polymerase

DNA STRUCTURE AND
FUNCTION
Chapter 10
Identification of the Genetic Material
Griffith’s Experiment
협막(capsule)
Griffith’s Experiment: What made R
strain into live S strain from dead one ?
그림 10.2
Infection of bacteriophage
Hershey-Chase Experiment
세균에 침투하는 것은 DNA이며, 이것이 파지 자손을 증식시키는
유전물질임을 증명 (Sulfur는 외피단백질의 원소가 되며, 인은 중핵
DNA의 원소가 됨)
표 10.1
A. DNA Structure
DNA is a nucleic acid composed of
nucleotide monomers.
DNA nucleotide consists of:
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•
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one phosphate group
one deoxyribose sugar (5 carbon sugar)
one nitrogenous base (G, A, C or T)
DNA is a double-stranded helix
(Watson & Crick 1953).
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Sides of ladder
make up sugarphosphate
“backbone”.
Rungs (band) of
ladder composed
of base pairs
joined by
hydrogen bonds.
Pyrimidines (T & C) form hydrogen
bonds with purines (A & G).
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Thymine pairs with
Adenine, forming 2
hydrogen bonds.
Cytosine pairs with
Guanine, forming 3
hydrogen bonds.
DNA strands are antiparallel.
5’ to 3’ strand
3’ to 5’ strand
Numbering of strands
is based on position
of deoxyribose
sugars.
DNA is highly condensed.
•
•
DNA is wrapped
tightly around
proteins(neucleosome)
& folded.
DNA must unwind for
replication to occur.
B. DNA Replication
Process by which DNA is duplicated.
occurs during the S phase of Interphase
• is semiconservative (Meselson & Stahl)
Exp.
• N15/N15에서 부모세대 배양후 방사능 검사
• N14에서 1세대 배양 (대장균:약30분) 후 방사능 검사
• 1세대 세균을 N14에서 2세대 배양 후 방사능 검사
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•

LH
LL
HH
LH
Overview of DNA Replication:
Unreplicated DNA.
Strands “unzip” at
several points creating
replication forks.
Each strand serves as
template for
complementary
nucleotides to H-bond.
New nucleotides of
each daughter strand
are linked.
Steps in DNA Replication:
Helicase breaks hydrogen bonds.
Binding proteins stabilize strands;
prevent them from rejoining.
Primase makes an RNA primer.
Free nucleotides move in & H-bond;
DNA polymerase links nucleotides to
each other starting at primer &
working in the 5’ to 3’ direction.
DNA polymerase “proofreads” new
strand (replaces incorrect bases).
DNA replication is continuous on one
strand.
DNA replication is discontinuous on
other strand, producing Okazaki
fragments.
Ligase
Repair enzymes remove RNA primers;
Ligase connects Okazaki fragments.
Determine the base sequence of
daughter DNA replicated from the
following parental DNA strand.
parental DNA
daughter DNA
C T A G G T A C T
G A T C C A T G A
C. DNA Repair
UV radiation damages DNA by causing
thymine dimers to form.
DNA damage can be repaired by
photoreactivation or excision repair.
Photolyase is present and functional in prokaryotes, is present
in lower eukaryotes (as yeast) where it is thought to have a
minor role, and it has not been found in human cells.
However, many higher eukaryotes, including humans, possess a
homologous protein called cryptochrome that is involved in
light-sensitive regulatory activities such as modulating
circadian rhythms.
Photolyases are flavoproteins and contain two light-harvesting
cofactors. All photolyases contain the two-electron-reduced
FADH2; they are divided into two main classes based on the
second cofactor, which may be either the pterin
methenyltetrahydrofolate (MTHF) in folate photolyases or
the deazaflavin 8-hydroxy-7,8-didemethyl-5-deazariboflavin
(8-HDF) in deazaflavin photolyases.
(from Wikipedia)
1. Photoreactivation –
photolyase (enzyme) uses
light energy to split dimer.
2. Excision repair -
repair enzyme cuts
out damaged area;
DNA polymerase
inserts replacement
sequence & ligase
seals backbone.
그림 10.12
3. Mismatch repair - enzymes
proofread newly replicated DNA for
base mispairing & correct the error.
Faulty DNA repair results in
chromosome breaks & an increased
susceptibility to cancer.
Ex. Xeroderma pigmentosum (열성유전
색소건피증)
D. Comparison of DNA & RNA
A =T
G
C
퓨린:피리미딘
오페론:세균에서의 유전자발현
E. Transcription
Process by which a molecule of RNA is
synthesized that is complementary to
a specific sequence of DNA
• Occurs in the nucleus of eukaryotic
cells & cytoplasm of prokaryotic cells.
• Is regulated by operons (bacterial cells)
or transcription factors (multicellular
organisms).
• Involves 3 stages: initiation,
elongation & termination
1. Initiation
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RNA polymerase attaches to a promoter on
DNA strand.
Helicase unzips a short section of DNA.
Free RNA nucleotides move in & H-bond to
complementary bases on DNA template
strand.
2. Elongation
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RNA polymerase links RNA nucleotides
together in a 5’ to 3’ direction.
Growing RNA strand peels away from DNA
template.
3. Termination
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RNA polymerase detaches when it reaches a
terminator.
Completed RNA
molecule is released
from DNA template.
Usually, several copies of RNA are made
at a time.
1
2
3
Determine the base sequence of RNA
transcribed from the following DNA
template strand.
DNA template C A G T A A G C C
RNA strand
G U C A U U C G G
Three major types of RNA are
transcribed.
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mRNA (messenger RNA) - encodes
genetic information from DNA & carries
it into the cytoplasm.
5’
3’
codon
Each three consecutive mRNA bases
forms a genetic code word (codon)
that codes for a particular amino acid.
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rRNA (ribosomal RNA) - associates
with proteins to form ribosomes.
large subunit
small subunit
Subunits are separate in the cytoplasm,
but join during protein synthesis
(translation).
•
tRNA (transfer RNA) - transports
specific amino acids to ribosome during
protein synthesis (translation).
Anticodon - specific
sequence of 3
nucleotides;
complementary to
an mRNA codon.
Amino acid
accepting end
Anticodon sequence determines the
specific amino acid that binds to tRNA.
The Genetic Code
Eukaryotic mRNA must be processed
before it exits nucleus & enters
cytoplasm.
• nucleotide cap
is added
• “poly A tail” is
added (a series
of 100-200
adenines)
• introns are
removed
F. Translation
Process by which an mRNA sequence is
translated into an amino acid
sequence (polypeptide/protein).
• Occurs in the cytoplasm of
eukaryotic & prokaryotic cells.
• Requires: mRNA, tRNAs, amino
acids & ribosomes.
• Involves 3 stages: initiation,
elongation & termination
The Genetic Code
1. Initiation
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Small ribosomal subunit binds to “start codon”
[AUG] on mRNA molecule.
AUG codon attracts initiator tRNA.
2. Elongation
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Large ribosomal subunit binds to small
subunit.
A second tRNA anticodon binds to the
next mRNA codon.
A peptide bond forms between the two
amino acids.
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Initiator tRNA is released.
Ribosome moves down mRNA by 1 codon.
A third tRNA anticodon binds to the
next mRNA codon.
A peptide bond forms between 2nd &
3rd amino acids.
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tRNAs continue to add amino acids;
polypeptide lengthens.
3. Termination
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Occurs when ribosome reaches an
mRNA stop codon (UGA, UAG or UAA).
Stop codons
do NOT
specify an
amino acid.
•
Last tRNA is released,
ribosomal subunits separate
& new polypeptide/protein is
released.
Usually, several copies of the
polypeptide/protein are made at a time.
6
5
4
3
2
1
Some polypeptides must be altered
before they can function.
Determine the amino acid sequence
a ribosome would translate from
the following mRNA strand.
mRNA C A U G G C U C A A U G A
Met
Ala
Gln
STOP
Review: Genetic information flows in cell
from DNA  RNA  protein.
Each gene on DNA codes for production
of a specific polypeptide/amino acid.
G. Mutation
A physical change in the nucleotide
sequence of DNA.
• May not affect phenotype (silent
mutation).
• Can affect somatic cells (somatic
mutation) or sex cells (germinal
mutation).
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Can form spontaneously or be
induced by a mutagen.
1. Point mutation - one DNA nucleotide
replaces another
• missense mutation - point mutation
that changes a codon to specify a
different amino acid.
Ex. sickle cell disease
nonsense mutation - point mutation
that changes an amino acid-specifying
codon into a stop codon.
2. Frameshift mutation - the insertion
or deletion of DNA nucleotides; results
in disruption of the reading frame.
Ex. cystic fibrosis (낭포성 섬유증)
3. Expanding repeat - the # of copies
of a 3 or 4 nucleotide sequence
increases over several generations.
Ex. myotonic dystrophy (근강직성
디스트로피증 )
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표 11. 6
표 11. 5
Natural protection against
mutation
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DNA proofreading
DNA repair
checking RNAs as they are
made
eliminating malformed proteins
genetic code
Protection in genetic code:
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synonymous codons (동의 코돈)
encode same amino acid, 3rd
position differs
mutation in 2nd codon position
changes specification to similar
amino acid
mutation affects phenotype only
if it alters protein’s function
p.201 그림 10B