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Various types of machinery are needed to
adjust the structure of chromatin rapidly
Chromatin-remodeling complexescan decondensechromatin
A molecular motor Needed for both replication
and gene expression. Hence, chromatin structure
is dynamic.
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Covalent modifications of histones regulate
chromatin structure (histone code) These modifications function, in part, by recruiting other
chromatin-remodeling complexes.*
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Covalent modifications of histones regulate
chromatin structure (histone code) These modifications function in part by recruiting other
chromatin-remodeling complexes.
Can also modify core
histones to help alter
chromatin (e.g., HATs)Still learning what
the codesmean
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Interphase chromosomes contain both
condensed and more extended chromatin Euchromatinmore extended, higher gene expression Heterochromatincondensed, low gene expression ~10% of interphase chromatin
Located around centromere and telomeres There are heterochromatin-specific proteins
Heterochromatin can
spread to coding regions
and silencethe gene.Barrier DNA prevents
heterochromatin spread
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Figure 5-29 Essential Cell Biology ( Garland Science 2010)
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Maternal Gene SilencingOne must be silenced,
so that expression in
females and males is
similar.Selection appears to
be randomSilenced X is passedalong the cell lineageFemales are Mosaics
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Chromatin structure can be inherited
(epigenetic inheritance)
Specific histone modifications are passed on. DNA methylation patterns are passed on. This process occurs during differentiation of cells (committed)
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Chapter 6DNA Replication, Repair, and
Recombination
EssentialCell Biology
Third Edition
Copyright Garland Science 2010
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Template-directed DNA Synthesis *
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Base-paring enables DNA replication
new strand
old strand
Watson-Crick base pairing; A-T and G-C
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How to distinguish between these 3 possibilities ?
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Meselson and Stahl Experiment
~ 1.2 % heavier
*14N15N
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14
N
15N
Meselson and Stahl Experiment
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14N
15N
Meselson and Stahl Experiment
(~ 1 generation)
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How to distinguish between these 3 possibilities ?
14
N
15N
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14N
15N
Meselson and Stahl Experiment
showed DNA replication is NOT conservative *
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How to distinguish between these 2 possibilities ?
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How to distinguish between these 2 possibilities ?
1
223 3
numberof
generations
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Meselson & Stahl (1958)
Meselson and Stahl Experiment
showed DNA replication IS semiconservative
3rd2nd1st
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DNA replication happens during S Phase *
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DNA replication begins at replication origins
Need to break H-bonds
Typically have veryspecific sequencesA-T rich
A-T rich
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Origin of Replication : Bacteria vs Human
Bacteria (~6 million bps) have single origin of replication intheir circular genome.
Human (~3 billion bps) have ~10,000 origins. Why so many origins?
bacteria human
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DNA synthesis starts at replication forks
Y-shaped structures (replication forks)
Bidirectional
S-Phase
Chromatin
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DNA polymerase is a core component of the
replication machinery
Synthesizes new DNA using one of parental strandsas template.
Catalyzes the addition of nucleotides to the 3-endof a growing DNA strand by forming
phosphodiester bonds between the 3hydroxyl
group and the 5-phosphate group of the incoming
nucleotide. The nucleotides come initially as high energy
nucleoside triphosphates (also a nucleotide). provides the energy for polymerization.
Thus,DNA is synthesized in the 5-to-3direction.
DNA polymerase remains bound to template strandand slides along the DNA.
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Figure 6-10 Essential Cell Biology ( Garland Science 2010)
Must have 3hydroxyl
to elongate the strand !!
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Figure 3-40 Essential Cell Biology ( Garland Science 2010)
ATP hydrolyzed to AMP can be used
if more energy is needed
!G0= 32.2 kJ/mol
!G0
= 33.5 kJ/molinorganic
pyrophosphatase
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DNA polymerase is self-correcting
Makes only ~1 error every 107bps. This is beyond the predicted accuracy.
DNA polymerase has 3-to-5proofreadingactivity. Monitors new base-pairing. Can correct mistake using a nuclease
that cleaves the phosphodiester
backbone.
Occurs during DNA synthesis.
Polymerization and proofreading are
carried out by 2 different domains of
DNA polymerase.
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Proofreading corrections are possible
only if DNA is synthesized 5-to-3
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The DNA replication fork is asymmetrical
Solution: DNA polymerase uses backstitchingto piece together short
strands of DNA called Okazaki fragments.
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Answer: DNA polymerase uses backstitchingto piecetogether short strands of DNA called Okazaki fragments.
Thus, this lagging strandis made discontinuously. These
short fragments are subsequently joined together.*
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Short RNAs are primers for DNA synthesis
Q: Since DNA pol (polymerase) can only join a nucleotide to abase-paired nucleotide in DNA, how does it start?A: It needs a primer nucleotide sequence.This priming is done by the enzymeprimase. This enzyme does
not need a base-paired end.Primase makes short stretches of RNA (~10 bps)!primersfor
DNA synthesis.
Primase is an example of anRNA polymerase-an enzyme
that makes RNA using a DNA template. This RNA/DNA
duplex is known as a heterodimer.
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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 anucleasethat recognizes the RNA/DNA
heterodimer. ADNA repair polymerase with
proofreading then fills in the gap (end ofOkazaki is primer).
The completed fragments are finallyjoined/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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Lagging strand cannot be completed
Must have a base-paired residue
with a 3hydroxyl to be
synthesized by the DNA
polymerase
Primase 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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Figure 6-18 Essential Cell Biology ( Garland Science 2010)
"
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Failure to repair DNA mistakes can have
serious consequences
Can lead to permanent changes in the DNAmutations.
Ex. Sickle Cell Anemia
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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 ofmutations 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 6-21b Essential Cell Biology ( Garland Science 2010)
BAD
WORSE
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Figure 6-21c Essential Cell Biology ( Garland Science 2010)
GOOD
GOOD
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DNA mismatch repair
Distorts dsDNA; hence
can be recognized as
different
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Figure 6-23 Essential Cell Biology ( Garland Science 2010)
Spontaneous events that compromise DNA integrity*
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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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D d DNA i i lf
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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 the
two strands. Proteins (nucleases) involved in Step 1
vary 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*
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Homologous recombinationduring meiosis
During meiosis, chromosomal crossovers lead tothe exchange of genetic information.
During meiosis recombination preferentially occurs
between maternal and paternal chromosomes rather
than between newly replicated, identical DNA
strands like when HR repairs dsDNA breaks.
(meiosis-specific proteins)
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Gene Conversion Crossover
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Barbara McClintock(1902 1992)
Discovery of Genetic TranspositionJumping GenesTransposons
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M bil G i El (T )
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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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M bil G i El (T )
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Mobile Genetic Elements (Transposons)
inverted repeats5---GACTGCGCAGTC---3
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Mobile Genetic Elements encode the
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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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Human genome contains 2 major
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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 relativelyrecently.
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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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Vi th lti t bil DNA
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Figure 6-37 Essential Cell Biology ( Garland Science 2010)
Viruses: the ultimate mobile DNA
Vi th lti t bil 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.
Vi a d th h t ll a hi
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Viruses commandeer the host cell s machinery
This lysis can cause an immune response.
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Retroviruses make DNA from an RNA template
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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
e.g., HIV*
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