Download Review of Advanced DNA Structure and Function PPT

Advanced DNA
Structure and
Function
Review
DNA Replication,
Repair, &
Recombination
DNA Replication
DNA serves as a template for its own duplication by way of
complementary base pairing
DNA replication is thus semiconservative: each new DNA
molecule is composed of one old strand & one new strand
Messelson-Stahl Experiment
• Cells were grown for
many generations in
15N.
• Cells transferred to
medium w/ 14N only.
Analyze DNA density
after first generation.
• Continue to grow cells
for a 2nd generation &
analyze DNA density.
DNA Replication is
Semiconservative
DNA Replication Begins at
Replication Origins
Replication Origin Recognition
Complex
DNA Replication
• The incoming
monomer
carries the
energy for its
own addition
• New strand
grows in the 5’
to 3’ direction
DNA Polymerase
DNA Replication
•
•
•
•
•
DNA Helicase
DNA Primase
DNA Polymerase
Nuclease
Repair DNA
Polymerase
• DNA Ligase
• Binding Proteins
• DNA
Topoisomerases
Helicase is an ATP binding
Protein
Bacteria
Primers are ~10 nucleotides
long. Start every 100-200
nucleotides.
DNA pol 
forms complex
w/ primase;
then further
extension is
taken over by
DNA pol .
Mammalian
Replication of the Ends of
Eucaryotic Chromosomes
• Telomerase
• RNA template is part of
enzyme structure
• In humans the sequence
is GGGGTTA
Blackburn, Greider, & Szostak
DNA Replication
• DNA Polymerase has proof
reading capabilities.
• “good” base pairing between
incoming nucleotide &
parental strand increases the
affinity of the enzyme
• Absolute requirement for a 3’
end
– Incorrect base pairing at the 3’
end is an ineffective substrate
– 3’ to 5’ exonuclease activity will
remove incorrect base
Mismatch Repair
• For Errors missed by DNA
Polymerase proofreading
• Strand Directed
• Enzymes “look” for
distortions in the DNA strand
• Remove preferentially newly
synthesized strand
– Unmethylated GATC sequences
– Nicked DNA
Complementary Base Pairing,
DNA Polymerase Proofreading,
Mismatch Repair
MISTAKE RATE = 1/109
(ERROR PER NUCLEOTIDE COPIED)
Histone Reassembly on new DNA
• DNA polymerase can traverse
through parental nucleosomes
without displacing them
• Old Histones are distributed to
daughter strands
• More Histones must be
synthesized
• Occurs specifically in S phase
• Histones loaded by chaperone
proteins
• Nucleosome assembly protein
• Chromatin assembly factor
• Any Histone covalent
modification is then re-established
Review
• Origin of Replication Initiated – this is controlled by cell cycle control
proteins which phosphorylate proteins preloaded at O.R.s. Helicase is
part of the complex.
• Helicase is activated; opens helix
• DNA primase produces primers
• DNA polymerase extends primers (5 3)
• Nucleases remove primers
• Ligase seals nicks
• Nucleosomal histones are synthesized and assemble after replication
fork moves on
• Telomerase completes ends of linear chromosomes
• Topoisomerase relieve supercoiling by creating breaks in the strands
of DNA outside of the fork and allowing the strands to swivel.
DNA Repair
Mutation
Germ Cells
Somatic Cells
Mismatch Repair
Damage Repair
Low Mutations Rates Are Necessary
for Life as We Know It
How Chemically Modified Nucleotides
Can Produce Mutations
Depurination & Deamination
• Most frequent spontaneous chemical reactions
that can create serious DNA Damage
UV Damage to DNA
xeroderma pigmentosum
Summary of DNA Spontaneous
Mutations Requiring Repair
Oxidative damage
Hydrolysis
Uncontrolled methylation
DNA
Damage
Repair
Mechanism
Nuclease (many types)
Repair Polymerase
Ligase
Two Major Pathways
Double Stranded Break Repair
• Nonhomologous end joining
– Degradation of ends and
ligation
– Loss of nucleotides
– Common in mammalian
somatic cells
• Homologous Recombination
– Seen during S phase and
G2 of eukaryotic cells
– Seen during DNA
replication of prokaryotic
cells
Homologous
Recombination
• Genetic exchange takes place
between a pair of homologous DNA
sequences.
• Usually no sequence alteration
occurs.
• Accurate repair of double strand
breaks caused by radiation, toxins, or
messed up replication forks.
Stalled Replication Fork
Double stranded
break caused by
radiation or toxin
repaired using a
sister chromatid
in S or G2 of cell
cycle as in a
eukaryotic cell.
Crossing Over of Meiosis
• Barbara McClintock & graduate student,
Harriet Creighton, first described crossing over
in corn (1929)
Crossing Over is Homologous
Recombination
• Homologous Recombination
– Base pairing cannot occur
between two intact DNA
molecules
– 1st double strand break occurs
(endonuclease)
– 2nd limited degradation
(exonuclease)
– 3rd pairing occurs with
homologous chromosome
(RecA protein; Rad
51+accessory proteins)
– Finally branch migration and
resolution
Lack of Rad51 will kill a cell; mutated
accessory proteins that control or assist
Rad51 can lead to cancer. Eg. Brca1 & 2
Figure 5-64 Molecular Biology of the Cell (© Garland Science 2008)
Holliday Junctions
This most
often
This less
often
Homologous Recombination
• Allows organism to repair DNA
• Required for accurate chromosomes
segregation during meiosis
• Creates new combination of alleles
DNA Recombination
•
•
•
•
•
•
Nonhomologous Recombination (Site
Specific Recombination) involving Mobile
Genetic Elements
Allows DNA exchange between DNA that
are dissimilar in sequence.
Mobile genetic elements vary in size (few
100 to 1000’s of bp)
Relics of mobile geneteic elements can
occupy large fxn of genome (Eg. >45%
human genome
Can impact gene sequences; responsible for
important evolutionary changes in genomes
Barbara McClintock first described
transposition in 1940’s; it took molecular
biology revolution for rest of the scientific
community to grasp the concept.
Won the 1983 Nobel Prize in
Physiology & Medicine
DNA Recombination
• Mobile Genetic Elements
can move by a process
called: Site Specific
Recombination
Examples of DNA only
transposons from bacteria
– DNA only transposons
– Retro-transposons
– Viruses
Transposase – the enzyme necessary to conduct
the DNA breakage & joining reactions needed for
the transposable element to move.
DNA Recombination
• Mobile Genetic Elements
can move by a process
called: Site Specific
Recombination
– DNA only Transposons
– Retro-transposons
– Viruses
This will have to be repaired – either by the double stranded break repair
mechanisms discussed earlier or an end joining mechanism that may alter the
original DNA sequences that flanked the transposon.
DNA Recombination
• Mobile Genetic Elements
can move by a process
called: Site Specific
Recombination
– DNA only Transposons
– Retro-transposons
– Viruses
Reverse transcriptase can
make dsDNA from RNA
Retroviral-Like
Retrotransposition
Nonretroviral Retrotransposition
DNA Recombination
• Mobile Genetic Elements
can move by a process
called: Site Specific
Recombination
– DNA only Transposons
– Retro-transposons
– Viruses
RTN
Reaction Catalyzed by Liagase
Procaryotic & Eucaryotic DNA Polymerase
Enzyme
Direction of Synthesis
Exonuclease Activity
Probable Function
RTN
Procaryotic
Polymerase I
5’  3’
5’  3’
3’  5’
gap filling after primer
removal; DNA repair
Polymerase II
5’  3’
3’  5’
gap filling after primer
removal; DNA repair
Polymerase III
5’  3’
5’  3’
3’  5’
Primary replication enzyme
Eucaryotic
Polymerase α
5’  3’
none*
Primary replication enzyme
(with polymerase δ); DNA
repair
Polymerase β
Polymerase γ
5’  3’
5’  3’
none*
3’5’
DNA repair
Primary replication enzyme
of mitochondria
Polymerase δ
5’  3’
3’5’
Primary replication enzyme
(with polymerase α)
Polyerase ε
5’  3’
3’5’
DNA repair
Transcription
From DNA to Protein
I. From DNA to RNA
• Transcription
• DNA used as a template to
form a complementary
RNA molecule
• Occurs in the nucleus of
eukaryotic cells; while
translation takes place in
the cytosol
I. From DNA to RNA
• Transcription
• DNA used as a template to form
a complementary RNA molecule
• Occurs in the nucleus of
eukaryotic cells; while
translation takes place in the
cytosol.
• RNA has ribose instead of
deoxyribose
• RNA has Uracil instead of
thymine
RNA Molecules Fold into
Complicated Shapes
Cells Make Several Types of RNA
PROKARYOTIC
TRANSCRIPTION
DNA to RNA
• RNA Polymerase binds to
Promotor
• Important point for gene
regulatory processes
• RNA polymerase forms a
polymer of ribonucleotides
complementary to the gene
sequence
• Terminator sequence results
in dissociation of RNA
polymerase from the DNA
Prokaryotic Sigma Factor
Basic Mechanism of Transcription is Similar
A FEW DIFFERENCES TO NOTE
Eukaryotic Transcription &
Translation are Separated
• Transcription in
cytosol of bacteria
but in nucleus of
eukaryotes
• Processing occurs in
eukaryotes
Processing Involves Intron Removal
• Eukaryotic Genes contained intervening sequences
(INTRONS)
• Eukaryotic primary transcript is processed in the
nucleus
DNA to RNA
• Eukaryotic Transcripts
undergo processing prior
to leaving the nucleus.
• For mRNA the
processing steps are:
– Addition of a 5’ CAP
– Removal of Introns
– Addition of a Poly A Tail
Bacterial Genes Are Often
Polycistronic
More than 1 Polymerase
• DNA is transcribed by
RNA Polymerase
• RNA Polymerase is a large
protein complex
• 1 RNA Polymerase in
prokaryotes
• 3 RNA Polymerases in
eukaryotes
Eukaryotic
General Transcription Factors
• Eukaryotic RNA Pol.
cannot begin transcription
on its own
• Assists binding
• TFIID binds TATA Box
• TFIIH phosphorylates the
RNA Pol
• Assembly of RNA
modification enzymes
EUKARYOTIC
TRANSCRIPTION
Figure 6-16 (part 1 of 3) Molecular Biology of the Cell (© Garland Science 2008)
Figure 6-16 (part 2 of 3) Molecular Biology of the Cell (© Garland Science 2008)
Figure 6-16 (part 3 of 3) Molecular Biology of the Cell (© Garland Science 2008)
Figure 6-16 Molecular Biology of the Cell (© Garland Science 2008)
Capping Factors = 3 enzymes
acting together (a phosphatase,
guanyl transferase, and a
methyl transferease)
phosphatase
Guanyl
transferase
Methyl
transferase
DNA to RNA
• 5’ Cap – a unique 5’
to 5’ linkage between
a methylated
guanosine and the 5’
end of the mRNA
Figure 6-8a Molecular Biology of the Cell (© Garland Science 2008)
DNA to RNA
• Removal of Introns
and Splicing of
Exons together
• Small nuclear
ribonucleoproteins
(snRNPs)
Although ATP
hydrolysis isn’t req’d
for RNA splicing per
se, it is req’d for
necessary splicesome
rearrangements
DNA to RNA
• Removal of Introns
and Splicing of
Exons together
• Small nuclear
ribonucleoproteins
(snRNPs)
• Alternate splicing
patterns can be seen
for many genes
DNA to RNA
• Polyadenylation –
addition of a few
hundred
adenosines to the
3’ end of the
mRNA
CPSF =
cleavage &
polyadenylati
on specificity
factor
CstF =
cleavage
stimulation
factor
Figure 6-38 Molecular Biology of the Cell (© Garland Science 2008)
Figure 6-38 (part 1 of 3) Molecular Biology of the Cell (© Garland Science 2008)
Figure 6-38 (part 2 of 3) Molecular Biology of the Cell (© Garland Science 2008)
Figure 6-38 (part 3 of 3) Molecular Biology of the Cell (© Garland Science 2008)
CBC = cap binding complex
EJC = exon junction complex
hnRNP = heterogenous nuclear ribonuclear proteins
Figure 6-39a Molecular Biology of the Cell (© Garland Science 2008)
Figure 6-39b Molecular Biology of the Cell (© Garland Science 2008)
DNA to RNA
• Eucaryotic Transcripts
undergo processing prior
to leaving the nucleus.
• For mRNA the
processing steps are:
– Addition of a 5’ CAP
– Removal of Introns
– Addition of a Poly A Tail
R.D.
Kornberg
2006 Nobel
Prize
DNA to Protein
• Orientation of the polymerase determines
which side of the DNA is used as the template
Transcription
From RNA
to Protein
Occurs in
Cytoplasm
Requires: mRNA,
amino acyltRNA’s, and
ribosomes
The Genetic Code
• mRNA – the sequence of nucleotides that
determines a sequence of amino acids
• 4 nucleotides
20 amino acids
• Group of 3 nucleotides = a codon
– (George Gamow)
1968 Nobel Prize to Nirenberg, Khorana and Holley
The Genetic Code
• How many possible combinations?
• 43 = 64 possible codons
• Now we have more codons than amino acids (i.e.
Genetic code is redundant or “degenerate”)
• Genetic code is universal (i.e. it has been highly
conserved evolutionarily)
Start & Stop Codons
In theory there would be 3
possible reading frames of
any mRNA
But …
the Start Codon establishes
the reading frame by
initiating protein synthesis
Protein Synthesis/Translation
• Codons don’t directly
bind to amino acids
• We need an adaptor
molecule (Francis
Crick 1955) --- tRNA
• Specific Enzymes
couple each amino
acid to its appropriate
tRNA molecule
Protein Synthesis/Translation
• Amino Acyl tRNA
synthetases
• 20 different versions
• Coupling reaction
generates an amino
acyl-tRNA
• Requires two high
energy P bonds
Protein Synthesis/Translation
• Most cells don’t have 61
tRNAs.
• Number varies but is typically
less than 61.
• Wobble base pairing =
nonstandard base pairing
between 3rd base of codon &
corresponding base on
anticodon
• Often U or C in 3rd position of
codon can pair with a G in
anticodon.
• Inosine can also occupy wobble
position in anticodon
Protein Synthesis/Translation
• Protein synthesis proceeds
on Ribosomes
• Small ribosomal subunit
binds to the 5’ end of the
mRNA and initiates the
process
• mRNA is read 5’ to 3’
• Protein is synthesized from
amino end to carboxy end
Initiation -- Procaryotes
Polycistronic
Initiation – Eucaryotes
Small subunit
Initiation Factors
Met
tRNA
i
Elongation Factor – 2
for translocation
(called EF-G in procaryotes)
Elongation
Peptidyl Transferase
Activity is a
Ribozyme
Elongation Factor-1
(called EF-tu in procaryotes)
Termination
Release Factor
t 1/2 of mRNA ~30
min.; gradual poly-A
shortening OR poly-A
removal
Molecular
Chaperones (hsp 60
& hsp 70)
Ubiquitin &
Proteosomes
rtn
DNA Learning Center
3-D Animation Library
http://www.dnalc.org/resources/3d/index.html
END