Rea Lec 9 DNA Replication
Transcript of Rea Lec 9 DNA Replication
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On the lagging strand, DNA is made in fragments
On the leading strand, only one initialprimer is needed. On the lagging strand, many primers
are needed. The RNA primers are removed by a
nucleasethat recognizes the RNA/DNA
heterodimer. ADNA repair polymerase with
proofreading then fills in the gap (end of
Okazaki is primer). The completed fragments are finally
joined/sealed byDNA ligase.
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DNA replication requires the coordination of
many proteins to form the replication machine
1. Need to unzip DNA-helicase (uses ATP)2. DNA polymerase3. Sliding clamp-keeps DNA pol on DNA.
Putting this on requires another protein-theclamp loader (uses ATP).
4. Need to stabilize ssDNA so it doesntrehybridize and keep it elongated-single-
strand binding protein (SSBPs)5. Primase, a nuclease (not shown here), DNA
repair pol, DNA ligase{For Lagging Strand
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DNA replication requires the coordination of
many proteins to form the replication machine
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DNA replication requires the coordination of
many proteins to form the replication machine
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DNA replication requires the coordination of
many proteins to form the replication machine
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DNA replication requires the coordination of
many proteins to form the replication machine *
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Telomerase replicates the ends of
eukaryotic chromosomes
Problem: DNA polymerase cannotsynthesize DNA in the 3-to-5direction.And, at the ends of chromosomes there is no place
to lay down an RNA primer. How are telomeres replicated? Solution: Eukaryotes have special
repetitive DNA sequences in theirtelomeres that recruit telomerase. Telomerase is part protein and part
RNA. It recognizes the repeats and
adds more repeats every time acell replicates its DNA.
Telomeres also identify ends ofchromosomes rather than dsDNA
breaksTelomerase linked to both
cancer and aging.
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Lagging strand cannot be completed
Must have a base-paired residue
with a 3hydroxyl to be
synthesized by the DNA
polymerasePrimase requires ~ 20 base pairs to
generate a 10 base pair primer
At some point, there is not enough
room left on the template strand
for the primase
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A DNA mismatch repair system removes
replication errors that escape DNA
polymerase proofreading DNA mismatch repairthe backup system
Fixes DNA mismatchesleft behind by replication machine. Pretty effective (>99%), but not perfect!
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Because germline mutations result in an entire
organism having mutation, protecting the
germ cells from mutations is critical Germ cellsthe reproduction cells = sperm and egg (ex. genetic diseases like SCA) Somatic cellsevery other cell in
your body (ex. cancer*)Due largely to the accumulation of
mutations over time. Anything that
speeds up this process could be
disastrous (ex. Mutation or deletion of
DNA repair enzyme).
colon
cancer in
women
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Mismatches must be repaired properly to
avoid mutations
bad worse good
In eukaryotes, still not known how DNA repair machinery tellsthe difference between the 2 strands. New DNA might be nicked
(ss breaks).
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Figure 6-21a Essential Cell Biology ( Garland Science 2010)
BAD*
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Figure 6-21c Essential Cell Biology ( Garland Science 2010)
GOOD
GOOD
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Figure 6-21b Essential Cell Biology ( Garland Science 2010)
BAD
WORSE
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Mismatches must be repaired properly to
avoid mutations
bad worse good
In eukaryotes, still not known how DNA repair machinery tellsthe difference between the 2 strands. New DNA might be nicked
(ss breaks).
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DNA mismatch repair
Distorts dsDNA; hencecan be recognized as
different
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the newly
synthesized
strand
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Spontaneous events that compromise DNA integrity*
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Spontaneous events that compromise DNA integrity*
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If not fixed, can lead to mutations
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Figure 6-24 Essential Cell Biology ( Garland Science 2010)
Thymine Dimers can form as consequence of UV radiation
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Damaged DNA can repair itself
using its backupcopy
Can use complementary strand as template. Since most DNA damage creates strange
looking structures, easy to differentiate thetwo strands.
Proteins (nucleases) involved in Step 1vary with different types of DNA damage.
Base Excision Repair (BER) System
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What happens when both strands
of DNA are damaged?
Can happen from ionizingradiation, replication fork errors,
various chemicals and
metabolites, etc.
Nonhomologous end-joining(NHEJ) is the most common
mechanism to repair dsDNA
breaks in somatic cells. Usually OK since most ofgenome non-coding.
Quick and dirty.
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Homologous Recombination
Can produce error-free repairs. Involves using entirely separate DNA
duplex(ex sister chromatids) to fix
dsDNA break. Also used extensively to produce genetic
diversity during meiosis (swapping between
maternal and paternal chromosomes).
Requires extensive stretches of sequencesimilarity (homology). But doesnt have to
be absolutely perfect homology.
Donor DNA needs to be in closeproximity following dsDNA break.
Versatile DNA repair mechanism. Highlyconserved.
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Homologous Recombination*
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Homologous recombinationduring meiosis
more common less common
If heteroduplex has anymismatches, DNA can
undergo mismatch repair.
Can lead to 2 differenttypes of recombination.
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Gene Conversion
Crossover
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Barbara McClintock(1902 1992)
Discovery of Genetic TranspositionJumping GenesTransposons
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Mobile Genetic Elements (Transposons)
jumping genes (molecular parasites ?) Short specialized sequences of DNA that can move throughout a
cells genome. Can carry other genes. Responsible for much more rapid evolutionary genetic changes. Typically affect only that cell and its descendants.
Can be major cause ofantibiotic resistant bacteria.
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Mobile Genetic Elements (Transposons)
inverted repeats5---GACTGCGCAGTC---3
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Mobile Genetic Elements encode the
components they need for movement
Unlike HR, dont require sequence homology. Contains
(1) Gene for transposase(catalyzes the movement of that element
via specialized recombination)(2) DNA sequences that are recognized by its transposase.
Nearly half of human genomeis occupied by millions of
copies of various mobile
genetic elements!!
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Figure 6-33 Essential Cell Biology ( Garland Science 2010)
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Human genome contains 2 major
families of transposable elements
1. DNA-only transposons2. Retrotransposons
Uses RNA intermediate Unique to eukaryotes Most common type
L1 element (LINE-1); 15% human genome Alu sequence; ~1 million copies in our
genome; dont encode their own reverse
transcriptase Both proliferated in primates relatively
recently.
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AluSequence Distribution
Arthrobacter luteus restriction endonuclease~ 300 bps
Formed from the 7SL RNA component
of the Signal Recognition Particle
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Figure 6-37 Essential Cell Biology ( Garland Science 2010)
Viruses: the ultimate mobile DNA
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Viruses: the ultimate mobile DNA
Essentially strings of genes wrapped in a protein coat. Very small. Parasitesthey need to use cells machinery to replicate. Often lethal (ex lytic) to cell.
Retroviruses are found only in eukaryotic cells.
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Figure 6-38 Essential Cell Biology ( Garland Science 2010)
Retroviruses make DNA from an RNA template
using reverse transcriptase *
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Retroviruses make DNA from an RNA template
using reverse transcriptase
Latent phase;
virus can hide
for a long time
Lytic phase
Major drug target for AIDSsince unique to virus
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End
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Chapter 7
From DNA to Protein: How Cells
Read the Genome
EssentialCell Biology
Third Edition
Copyright Garland Science 2010
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Charles Robert Darwin Alfred Russell Wallace
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Francis Harry C. Crick James Dewey Watson
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The Central Dogma of Molecular Biology
Occurs in all cells frombacteria to humans.
One of the definingcharacteristics of living cells. Allows massive amplification
of signals from a single gene.
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Figure 7-8 Essential Cell Biology ( Garland Science 2010)
Caught in the Act
Actively Transcribing Vertebrate DNA
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The efficiency of gene expression
is quite variable
Translation efficiency, as well asRNA and protein stability vary
greatly among genes
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Structure of RNA
Differs from DNA in 2 significant ways:1. ribonucleotides vs deoxyribnucleotides2. uracil vs thymine
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RNA is typically single stranded
Because RNA is single stranded, intramolecular base pairingcan occur, resulting in elaborate secondary structure
This gives rise to diverse functionality (e.g., ribozymes,riboswitches, tRNA, rRNA, )
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RNA serves many functions
final product = RNA molecules
Gene expression refers to the biosynthesis of either
DNA-encoded protein, or non-coding RNA
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From an evolutionary
perspective, RNA may have been
the original, self-replicatingbiopolymer
Ribozymes may have developedthe ability to direct protein
synthesis
DNA is probably a relative
newcomer, usurping RNAs role
in information storage
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Transcription is the DNA-directed biosynthesis of a
single, complementary RNA molecule
RNA is much shorter than DNA. RNA polymerase carries out transcription. RNA polymerase does not need a primer. Many RNA polymerases can transcribe a
single gene at the same time.
Transcription does havesimilarities to replication.
As for DNA, RNA issynthesized in the 5to 3
direction.
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Figure 7-7 Essential Cell Biology ( Garland Science 2010)
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E k i i i diff f b i l
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Eukaryotic transcription differs from bacterial
transcription in a few ways
1. Bacteria have only 1 RNA pol. Eukaryotes have 3.
2. Eukaryotic RNA polymerases require a bunch of accessory proteinscalled the general transcription factors (GTFs) to initiate
transcription.3. Control mechanisms are more complex in eukaryotes in part because
genes are much further apart. This allows more sophisticated gene
regulation.4. Eukaryotic transcription has to deal with more compact chromatin
structure.
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B i l d h ifi
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Bacterialpromotersand terminatorshave specific
sequences recognized by RNA polymerase
The promoter orients RNA pol and tells it where to start and which way to go. All bacterial genes have promoter and terminator sequence similar to those shown
below.
C i f DNA ll RNA l *
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Certain sequences of DNA tell RNA polymerase
where to start (promoters) and stop (terminator/stop site)
In bacteria, the sigma
factor, a subunit of the
RNA pol, recognizes the
promoter.
This is a dynamic process.
In bacteria
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B i l T i i
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Figure 7-9 Essential Cell Biology ( Garland Science 2010)
Bacterial Transcription*
Ei h d f DNA l
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Either strand of DNA can act as a template,
but the promoter is asymmetrical
Th G l T i i F *
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The General Transcription Factors
Assemble on the promoter. Position RNA polymerase. Open DNA. Launch the RNA polymerase.
typically ~25 bp upstream
of start site; mostpromoters have this
transcription initiation
complex
Both opens DNA and
phosphorylates and
releases RNA pol from
initiation complex
TBP is a
subunit ofTFIID that
distorts
DNA,
forming
landmark
All components are then laterrecycled to be used again and
again
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Figure 7-12 (part 1 of 2) Essential Cell Biology ( Garland Science 2010)
~ 25 bp upstream
from transcription
start siteTBP distorts DNA
TFIIB provides scaffold
TFIIH separates strands
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TATA B Bi di P t i di t t th d bl h li
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Figure 7-13 Essential Cell Biology ( Garland Science 2010)
TATA Box Binding Protein distorts the double helix*
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Figure 7-12 (part 1 of 2) Essential Cell Biology ( Garland Science 2010)
~ 25 bp upstream
from transcription
start siteTBP distorts DNA
TFIIB provides scaffold
TFIIH separates strands
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Figure 7-12 (part 2 of 2) Essential Cell Biology ( Garland Science 2010)
TFIIF phosphorylates tail
domain of RNA pol II
. launches polymerase
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Control of transcription initiation
is the most common wayorganisms regulate control gene
expression.
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Eukaryotic RNAs must be processed and
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In eukaryotes, transcriptionoccurs in nucleus, translation
occurs in cytoplasm. The
exportof RNA occurs via
nuclear pore complexes in thenuclear envelope.Prior to nuclear export,RNA
processingoccurs as the RNAmolecule is being synthesized.
Eukaryotic RNAs must be processed and
exported to cytoplasm
RNA i
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Occurs as RNA is beingmade.
Processing machinery isrecruited to the
phosphorylated taildomain of the eukaryotic
RNA polymerase. Different types of processing
occurs depending of what
type of RNA is being made.
RNA processing *
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Figure 7-15 Essential Cell Biology ( Garland Science 2010)
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E k ti RNA i
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Addition of 5-caps and 3-polyadenylation tails (poly-A tails)
(also intron splicing) . 5-caps and poly-A tails:
1. Increase stability.2. Identifies the molecule as mRNA.3. Marks the mRNA as being
complete.
Eukaryotic mRNA processing
Usually gets trimmed back firstbefore few hundred Aadded.
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Introns in eukaryotic mRNA are
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Eukaryotic genes are often interrupted by noncodingsequences (introns). Need to remove/splice these introns out to
get finished/meaningful message. Exons-expressed sequences Introns-intervening/nonexpressed sequences
Introns in eukaryotic mRNA are
removed by RNA splicing
Splicing can happen in prokaryotes, but rarely does.
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Introns are removed by RNA splicing
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Occurs during transcription after 5-capping. Can occur before during or afteraddition of poly-A tail.
Involves cutting out introns and stitchingexons back together.
Introns are removed by RNA splicing
Unlike exons, most of intron sequence appearsto be unimportant. There are a few short
sequences at or near each intron end that act as
cues for removal.
carried our primarily by catalytic RNA
molecules (small nuclear RNAs; snRNAs)
coupled to a few proteins to form small nuclear
ribonucleoprotein particles (snRNPs)forming
the core of the Spliceosome.
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This process carried our primarily by catalytic RNA
molecules (small nuclear RNAs; snRNAs) coupled to a fewproteins to form small nuclear ribonucleoprotein particles
(snRNPs)forming the core of the Spliceosome.
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Figure 7-20 Essential Cell Biology ( Garland Science 2010)
snRNPs bind to specific
sequences at both ends of
the intron.The 2hydroxyl of a
conserved Aattacks 5
splice site forminglariat
3hydroxyl of first exon
attacks 3splice site,
knitting exons together
Lariat is degraded
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Introns are removed by RNA splicing
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Introns are removed by RNA splicing
Alternative splicing leads to greater protein diversity fromsingle gene. ~60% of human genes undergo alternative splicing. Could have helped speed evolution of eukaryotes
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