Welcome to My Molecular Biology Class

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Welcome to My Molecular Biology Class
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Molecular Biology of the Gene, 5/E --- Watson et
al. (2004)
Part I Chemistry and Genetics Part II
Maintenance of the Genome Part III Expression
of the Genome Part IV Regulation Part V Methods
3/22/05
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Part II Maintenance of the Genome
Dedicated to the structure of DNA and the
processes that propagate, maintain and alter it
from one cell generation to the next
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Ch 6 The structures of DNA and RNA Ch 7
Chromosomes, chromatins and the nucleosome Ch 8
The replication of DNA Ch 9 The mutability and
repair of DNA Ch 10 Homologous recombination at
the molecular level Ch 11 Site-specific
recombination and transposition of DNA
3/22/05
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The consequence of high rates of mutation

• Mutation in germ line (????)
• would destroy the species
• Mutation in soma (???) would destroy the
  individual.

Maintenance of the correctness of the DNA
sequence is definitely crucial for living
organisms. Keeping the error rate as low as 10-10
(page191) is very expensive.
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Build up a serious attitude to science!!!

• I absolutely do not agree with Waston et al. at
  the points described 3rd 4th paragraphs on page
  235
• What are the more reasonable explanations for the
  10-10 mutation frequency in living organisms?
• What are the evidences that such a low mutation
  rate can drive the evolvement of a new species if
  the cell changes are known harmful?

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• Listening to nature
• Mutation (??) is naturally avoided by repair of
  the errors and damages, suggesting it is
  generally not welcome in living organisms.
• Recombination (??) is good and naturally
  promoted it is responsible for diversity inside
  of species.
• Transposition (??) is different from mutation and
  recombination because (1) producing mechanism is
  different (2) no mechanism to correct it (3)
  existing in nature in a well-controlled manner
  (10-5). Not repaired but controlled.

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• Molecular Biology Course

3 teaching hours

• CHAPTER 9 The mutability and repair of DNA
  

• Replication errors and their repair
• DNA damage
• Repair of DNA damage

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Two important sources for mutation (unavoidable)

• Inaccuracy in DNA replication (10-7 is not
  accurate enough)
• Errors (??)
• Chemical damage to the genetic material
  (environment)
• Lesions (??,??arose from spontaneous damage)
• Damage (??,?? caused by chemical agents and
  radiation

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To repair an error or damage

• First, Detect the errors
• Second, Mend/repair the errors or lesions in a
  way to restore the original DNA sequence.

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Questions to be addressed

• How is the DNA mended rapidly enough to prevent
  errors from becoming set in the genetic material
  as mutation
• How does the cell distinguish the parental strand
  from the daughter strand in repairing replication
  errors

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• How does the cell restore the proper DNA sequence
  when the original sequence can no longer be read?
• How does the cell deal with lesions that block
  replication?

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CHAPTER 9 The mutability and repair of DNA
Topic 1 Replication errors and their repair

• How the replication errors are resulted?
• The nature of the replication errors is mismatch.
• 2. How mismatches are recognized and correctly
  repair?

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The nature of mutations
Replication errors and replication

• Point mutations
• Transitions (pyrimidine to pyrimidine, purine to
  purine)
• Transversions (pyrimidine to purine, purine to
  pyrimidine)

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Insertions Deletions Gross rearrangement of
chromosome.
These mutations might be caused by insertion by
transposon or by aberrant action of cellular
recombination processes.
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Rate of spontaneous mutation at any given site on
chromosomal ranges from 10-6 to 10-11 per round
of DNA replication, with some sites being
hotspot . Mutation-prone sequence in human
genome are repeats of simple di-, tri- or
tetranucleotide sequences, known as DNA
microsatellites (???DNA). These sequences (1) are
important in human genetics and disease, (2) hard
to be copied accurately and highly polymorphic in
the population.
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• How the replication errors are resulted?

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Each bases has its preferred tautomeric form (Ch
6)
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The strictness of the rules for Waston-Crick
pairing derives from the complementarity both of
shape and of hydrogen bonding properties between
adenine and thymine and between guanine and
cytosine.
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Some replication errors escape proofreading
Replication errors and replication
The 3-5 exonuclease activity of replisome only
improves the fidelity of DNA replication by a
factor of 100-fold. The misincorporated
nucleotide needs to be detected and replaced,
otherwise it will cause mutation (Fig. 9-2).
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Figure 9-2 Generation of Mutation
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2. How the replication errors are repaired?
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Mismatch repair removes errors that escape
proofreading
Replication errors and replication
Increase the accuracy of DNA synthesis for 2-3
orders of magnitudes. Two challenges (1)rapidly
find the mismatches/mispairs, (2) Accurately
correct the mismatch
Talking about the story of E. coli repair system
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• MutS scans the DNA, recognizing the mismatch from
  the distortion they cause in the DNA backbone
• MutS embraces the mismatch-containing DNA,
  inducing a pronounced kink in the DNA and a
  conformational change in MutS itself

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MutS is a dimer. One monomer interacts with the
mismatch specifically, and the other
nonspecifically.
DNA is kinked
Figure 9-4 Crystal structure of MutS
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• MutS-mismatch-containing DNA complex recruits
  MutL, MutL activates MutH, an enzyme causing an
  incision or nick on one strand near the site of
  the mismatch. Nicking is followed by the specific
  helicase (why?) (UrvD) and one of three
  exonucleases (why?).

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Helicase
Exonuclease,
DNA polymerase III
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• Detail 1 How does the E. coli mismatch repair
  system know which of the two mismatched
  nucleotide to replace?

The newly synthesized strand is not methylated by
Dam methylase in a few minutes after the
synthesis.
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Figure 9-5
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• Detail 2 Different exonucleases are used to
  remove ssDNA between the nick created by MutH and
  the mismatch.

Figure 9-6
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• Eukaryotic cells also repair mismatches and do so
  using homologs to MutS (MSH) and MutL (MLH). The
  underlying mechanisms are not the same and not
  well understood.

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CHAPTER 9 The mutability and repair of DNA
Topic 2 DNA dmage
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DNA undergoes damage spontaneously (???) from
hydrolysis (??) and deamination (??)
DNA damage
Resulted from the action of water
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Figure 9-7 Mutation due to hydrolytic damage
Deamination C?U
Hydrolysis creates apurinic deoxyribose
Deamination 5-mC ? T
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Vertebrate DNA frequently contains 5-methyl
cytosine in place of cytosine as a result of the
action of methyl transferase. This modified base
plays a role in the transcriptional silencing (Ch
17).

• The presence of U and apurinic deoxyribose in DNA
  resulted from hydrolytic reactions is regarded as
  unnatural, thus is easily be recognized and
  repaired.

Can 5-mC ? T lesion be repaired?
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DNA is damaged by alkylation (???), oxidation
(??) and radiation (??)
DNA damage
Alkylating chemical Nitrosamines (???)
Reactive oxygen species (O2-, H2O2, OH)
Figure 9-8 G modification
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O2- hyperoxide H2O2 Peroxide OH
hydroxyl
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Figure 9-9 Thymine dimer. UV induces a
cyclobutane (???) ring between adjacent T.
Radiation damage 1
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• Gamma radiation and X-rays (ionizing radiation)
  cause double-strand breaks and are particularly
  hazardous (hard to be repaired).

Radiation damage 2
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Mutations are also caused by base analogs (?????)
and intercalating agents (???)
DNA damage

• Base analogs similar enough to the normal bases
  to be processed by cells and incorporated into
  DNA during replication.
• But they base pair differently, leading to
  mispairing during replication.
• The most mutagenic base analog is 5-bromoUracil
  (5-BrU) (????).

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(No Transcript)
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????
?????
Figure 9-10a Base analogues
Figure 3-33 G-U pair
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• Intercalating agents are flat molecules
  containing several polycyclic rings that interact
  with the normal bases in DNA through hydrogen
  bonds and base stacking.

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????
??, ??
?????/???
Figure 9-10b Intercalating agents
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CHAPTER 9 The mutability and repair of DNA
Topic 3 Repair of DNA damage
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Two consequence of DNA damage
Repair of DNA damage

• Some damages, such as thymine dimer, nick or
  breaks in the DNA backbone, create impediments to
  replication or transcription
• Some damages creates altered bases that has no
  effect on replication but cause mispairing, which
  in turn can be converted to mutation.

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See Table 9-1 for summary
Mechanisms to repair a damage
Repair of DNA damage

• Direct reversal of DNA damage by
  photoreactivation (?????) and alkyltransferase
  (?????)
• Base excision repair (????)
• Nucleotide excision repair
• Recombination (DSB) repairs
• Translesion DNA synthesis

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Direct reversal of DNA damage
Repair of DNA damage
Error-free repair
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Figure 9-11
Photoreactivation
Monomerization of thymine dimers by DNA
photolyases in the presence of visible light.
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Figure 9-12
Methyltransferase
Removes the methyl group from the methylated
O6-methylguanine. The methyl group is
transferred to the protein itself, inactivating
the protein.
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Base Excision repair enzyme remove damaged bases
by a base-flipping mechanism
Repair of DNA damage

• Glycosylase
• Recognizes the damaged base
• Removes the damaged base
• AP endonulease exonulcease
• 3.Cleaves the abasic sugar-phosphate backbone
• Exonulcease/DNA polymerase/ligase
• 4. Works sequentially to complete the repair
  event.

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Figure 9-14 base-flipping recognition by
glycosylase
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Figure 9-13 removes the damaged base and repair
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Fail-safe systems (??????)
Figure 9-15
oxoGA repair. A glycosylase recognizes the
mispair and removes A. A fail-safe glycosylase
also removes T from TG mispairs, as if it knows
how T is produced.
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Nucleotide Excision repair enzymes cleave damaged
DNA on either side of the lesion
Repair of DNA damage

• Recognize distortions to the shape of the DNA
  double helix
• Remove a short single-stranded segment that
  includes the lesion.
• DNA polymerase/ligase fill in the gap.

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Figure 9-16
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Figure 9-17. Transcription-couple repair
nucleotide excision repair (NER) system is
capable of rescuing RNA polymerase that has been
arrested by the presence of lesions in the DNA
template
TFIIH
TFIIH is a transcription factor including XPA and
XPD (UvrB)
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Recombination repairs DNA breaks by retrieving
sequence information from undamaged DNA
Repair of DNA damage
Double-strand break (DSB) repair pathway Details
are in chapter 10
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Figure 10-4. Damage in the DNA template can lead
to DSB formation during replication
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FIGURE 10-3 DSB repair model for homologous
recombination
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Translesion DNA synthesis enables replication to
proceed across DNA damage

• Error-prone repair
• Occurs when the above repairs are not efficient
  enough so that a replicating polymerase
  encounters a lesion
• Translesion synthesis is also called a fail-safe
  or last resort mechanism.

Repair of DNA damage
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• Translesion synthesis is catalyzed by a
  specialized class of DNA polymerases that
  synthesize DNA directly across the damage site.
• Translesion polymerase is produced by cell in
  response to the DNA damage
• Translesion polymerases are expressed as part of
  the SOS response pathway.

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FIGURE 9-19 Crystal structure of a translesion
polymerase. A Y-family polymerase found in many
organisms.
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FIGURE 9-19 Translesion DNA synthesis in E. coli
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CHAPTER 9 The mutability and repair of DNA
Summary and key points
All the repair mechanisms (details) and the cause
of the corresponding DNA errors and DNA
damages Mismatch repair system DNA replication
errors Direct reversal of DNA damages utraviolet
(UV) irradiation induced thymine/pyrimidine
dimmers---photoactivation alkylation agents
caused O6-methylguanine---methyl
transferase. Base excision repair the base
damage by alkylation and oxidation Nucleotide
excision repair the distortion of the DNA double
helix by thymine dimmer or the bulky chemical
adduct (???) on a base. Recombination repair
double-strand breaks in DNA, errors encountered
by a replication fork. Translesion synthesis
allows the replication to proceed across DNA
damage at a cost of error-prone replication. A
different DNA polymerase is utilized.
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CHAPTER 9 The mutability and repair of DNA
Homework
Review the lecture