Welcome Each of
You to My
Molecular Biology
Class
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
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
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
CHAPTER 9,The mutability
and repair of DNA
?Molecular Biology Course
Different changes of DNA
-behavior re-address (行为矫正）
? Chapter 8,Mutation (突变 ) is bad
(death and unhealthy),which
needs to be repaired
? Chapter 9,Recombination (重组 )
is good (diversity in a species-
beautiful),which is promoted
? Chapter 10,Transposition (转座 )
is not bad,because it is not
repaired,(benefit?)
The consequence of high
rates of mutation
? Mutation in germ line (生殖细胞 )
would destroy the species
? Mutation in soma (体细胞 ) 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 is so expensive.
Be fair (公正 ) and serious
(严肃 ) to science
The points that I absolutely do not
agree with Waston et al.
1,Mutation is not good,it could not
be responsible for biodiversity,
2,Transposition is different from
mutation because (1) producing
mechanism is different; (2) no
mechanism to correct it; (2)
existing in nature in a well-
controlled manner (10-5),(物种起源 )
Two important sources
for mutation (unavoidable)
? Inaccuracy in DNA replication (10-7 is
not accurate enough)
Errors (错误 )
? Chemical damage to the genetic
material (environment)
Lesions (损害,伤害 arose from spontaneous
damage)
Damage (损害,伤害 caused by chemical
agents and radiation
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.
Questions to be addressed
1,How is the DNA mended rapidly
enough to prevent errors from
becoming set in the genetic
material as mutation
2,How does the cell distinguish
the parental strand from the
daughter strand in repairing
replication errors
3,How does the cell restore the
proper DNA sequence when the
original sequence can no longer
be read?
4,How does the cell deal with
lesions that block replication?
Topic 1,Replication
errors and their repair
CHAPTER 9 The mutability and repair of DNA
3/22/05
The nature of mutations
Point mutations,
1,Transitions (pyrimidine to
pyrimidine,purine to purine)
2,Transversions (pyrimidine to
purine,purine to pyrimidine)
Re
pli
ca
tio
n e
rro
rs
an
d r
ep
lic
ati
on
Insertions
Deletions
Gross rearrangement of chromosome,
These mutations might be
caused by insertion by
transposon or by aberrant
action of cellular recombination
processes.
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 (微卫星 DNA),
These sequences (1) are important in
human genetics and disease,(2) hard
to be copied accurately and highly
polymorphic in the population.
Some replication errors escape
proofreading
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.
Re
pli
ca
tio
n e
rro
rs
an
d r
ep
lic
ati
on
Figure 9-2 Generation of Mutation
Mismatch repair removes errors
that escape proofreading
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
Re
pli
ca
tio
n e
rro
rs
an
d r
ep
lic
ati
on Talking about the story of
E,coli repair system
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
Figure 9-4 Crystal structure of MutS
MutS is a dimer,
One monomer
interacts with
the mismatch
specifically,and
the other
nonspecifically,
DNA is kinked
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 (?) (UrvD) and one of
three exonucleases (?).
DNA polymerase III
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,
Figure 9-5
Detail 2,Different exonucleases are
used to remove ssDNA between
the nick created by MutH and the
mismatch,
Figure 9-6
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,
Topic 2,DNA dmage
CHAPTER 9 The mutability and repair of DNA
3/22/05
DNA undergoes damage
spontaneously (自发的 ) from
hydrolysis and deamination
Resulted from the action of water
DNA
damag
e
Figure 9-7,
Mutation due
to hydrolytic
damage
Deamination C?U
Hydrolysis creates apurinic deoxyribose
Deamination 5-mC ? T
The presence of U and apurinic
deoxyribose in DNA resulted from
hydrolytic reactions is regarded as
unnatural,thus is easily be
recognized and repaired,
Explaining why DNA contains T
instead of U (?)
Can 5-mC ? T lesion be repaired?
DNA is damaged by alkylation
(烷基化 ),oxidation (氧化 ) and
radiation (辐射 )DNA
damag
e
Figure 9-8 G modification
Nitrosamines (亚硝胺 )
Reactive oxygen species (O2-,H2O2,OH?)
Figure 9-9 Thymine dimer,UV induces
a cyclobutane (环丁烷 ) ring between
adjacent T.
Gamma radiation and X-rays
(ionizing radiation) cause double-
strand breaks and are particularly
hazardous (hard to be repaired).
Mutations are also caused by
base analogs and intercalating
agentsDNA
damag
e
?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 mistake during
replication.
?The most mutagenic base anolog
is 5-bromouracil.
?Intercalating agents are flat
molecules containing several
polycyclic rings that interact
with the normal bases in DNA
through hydrogen bonds and
base stacking.
Topic 3,Repair of DNA
damage
CHAPTER 9 The mutability and repair of DNA
3/22/05
Two consequence 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.
Rep
air
of
DNA
damag
e
?Direct reversal of DNA damage by
photoreactivation (光活化作用 ) and
alkyltransferase (烷基转移酶 )
?Base excision repair (切割修复 )
?Nucleotide excision repair
?Recombination (DSB) repairs
?Translesion DNA synthesis
Mechanisms to repair a damage
See Table 9-1 for summary
Rep
air
of
DNA
damag
e
Direct reversal of DNA damage
Error-free repair
Rep
air
of
DNA
damag
e
Photoreactivation
Figure 9-11
Monomerization of thymine dimers
by DNA photolyases in the presence
of visible light.
Methyltransferase
Removes the methyl group from
the methylated O6-methylguanine,
The methyl group is transferred to
the protein itself,inactivating the
protein.
Figure 9-12
Base Excision repair enzyme
remove damaged bases by a base-
flipping mechanismRep
air
of
DNA
damag
e
Glycosylase
? Recognizes the damaged base
? Removes the damaged base
AP endonulease & exonulcease
3.Cleaves the abasic sugars
Exonulcease/DNA
polymerase/ligase
4,Works sequentially to
complete the repair event.
Figure 9-14,
base-flipping
recognition
Figure 9-13,removes the damaged
base and repair
oxoG:A repair,A glycosylase recognizes the
mispair and removes A,A fail-safe
glycosylase also removes T from T:G
mispairs,as if it knows how T is produced?
Fail-safe systems (最后保险系统 )
Figure 9-15:
Nucleotide Excision repair
enzymes cleave damaged DNA on
either side of the lesionRep
air
of
DNA
damag
e
1.Recognize distortions to the
shape of the DNA double helix
2.Remove a short single-
stranded segment that
includes the lesion,
3.DNA polymerase/ligase fill in
the gap.
Figure 9-16**
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
Recombination repairs DNA breaks
by retrieving sequence information
from undamaged DNARep
air
of
DNA
damag
e
Double-strand break (DSB)
repair pathway
Details are in chapter 10
Figure 10-4,Damage
in the DNA template
can lead to DSB
formation during
replication
FIGURE 10-3 DSB repair model for
homologous recombination
Translesion DNA synthesis enables
replication to proceed across DNA
damageRep
air
of
DNA
damag
e
?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,
1.Translesion synthesis is
catalyzed by a specialized
class of DNA polymerases that
synthesize DNA directly
across the damage site,
2.Translesion polymerase is
produced by cell in response
to the DNA damage
3.Translesion polymerases are
expressed as part of the SOS
response pathway,
FIGURE 9-19 Crystal structure of a
translesion polymerase,A Y-family
polymerase found in many organisms,
FIGURE 9-19
Translesion DNA
synthesis in E,coli
重点
3/25/05
掌握 slides里的内容。
CHAPTER 9 The mutability and repair of DNA
Review the lecture
Homework
CHAPTER 9 The mutability and repair of DNA
You to My
Molecular Biology
Class
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
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
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
CHAPTER 9,The mutability
and repair of DNA
?Molecular Biology Course
Different changes of DNA
-behavior re-address (行为矫正）
? Chapter 8,Mutation (突变 ) is bad
(death and unhealthy),which
needs to be repaired
? Chapter 9,Recombination (重组 )
is good (diversity in a species-
beautiful),which is promoted
? Chapter 10,Transposition (转座 )
is not bad,because it is not
repaired,(benefit?)
The consequence of high
rates of mutation
? Mutation in germ line (生殖细胞 )
would destroy the species
? Mutation in soma (体细胞 ) 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 is so expensive.
Be fair (公正 ) and serious
(严肃 ) to science
The points that I absolutely do not
agree with Waston et al.
1,Mutation is not good,it could not
be responsible for biodiversity,
2,Transposition is different from
mutation because (1) producing
mechanism is different; (2) no
mechanism to correct it; (2)
existing in nature in a well-
controlled manner (10-5),(物种起源 )
Two important sources
for mutation (unavoidable)
? Inaccuracy in DNA replication (10-7 is
not accurate enough)
Errors (错误 )
? Chemical damage to the genetic
material (environment)
Lesions (损害,伤害 arose from spontaneous
damage)
Damage (损害,伤害 caused by chemical
agents and radiation
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.
Questions to be addressed
1,How is the DNA mended rapidly
enough to prevent errors from
becoming set in the genetic
material as mutation
2,How does the cell distinguish
the parental strand from the
daughter strand in repairing
replication errors
3,How does the cell restore the
proper DNA sequence when the
original sequence can no longer
be read?
4,How does the cell deal with
lesions that block replication?
Topic 1,Replication
errors and their repair
CHAPTER 9 The mutability and repair of DNA
3/22/05
The nature of mutations
Point mutations,
1,Transitions (pyrimidine to
pyrimidine,purine to purine)
2,Transversions (pyrimidine to
purine,purine to pyrimidine)
Re
pli
ca
tio
n e
rro
rs
an
d r
ep
lic
ati
on
Insertions
Deletions
Gross rearrangement of chromosome,
These mutations might be
caused by insertion by
transposon or by aberrant
action of cellular recombination
processes.
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 (微卫星 DNA),
These sequences (1) are important in
human genetics and disease,(2) hard
to be copied accurately and highly
polymorphic in the population.
Some replication errors escape
proofreading
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.
Re
pli
ca
tio
n e
rro
rs
an
d r
ep
lic
ati
on
Figure 9-2 Generation of Mutation
Mismatch repair removes errors
that escape proofreading
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
Re
pli
ca
tio
n e
rro
rs
an
d r
ep
lic
ati
on Talking about the story of
E,coli repair system
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
Figure 9-4 Crystal structure of MutS
MutS is a dimer,
One monomer
interacts with
the mismatch
specifically,and
the other
nonspecifically,
DNA is kinked
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 (?) (UrvD) and one of
three exonucleases (?).
DNA polymerase III
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,
Figure 9-5
Detail 2,Different exonucleases are
used to remove ssDNA between
the nick created by MutH and the
mismatch,
Figure 9-6
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,
Topic 2,DNA dmage
CHAPTER 9 The mutability and repair of DNA
3/22/05
DNA undergoes damage
spontaneously (自发的 ) from
hydrolysis and deamination
Resulted from the action of water
DNA
damag
e
Figure 9-7,
Mutation due
to hydrolytic
damage
Deamination C?U
Hydrolysis creates apurinic deoxyribose
Deamination 5-mC ? T
The presence of U and apurinic
deoxyribose in DNA resulted from
hydrolytic reactions is regarded as
unnatural,thus is easily be
recognized and repaired,
Explaining why DNA contains T
instead of U (?)
Can 5-mC ? T lesion be repaired?
DNA is damaged by alkylation
(烷基化 ),oxidation (氧化 ) and
radiation (辐射 )DNA
damag
e
Figure 9-8 G modification
Nitrosamines (亚硝胺 )
Reactive oxygen species (O2-,H2O2,OH?)
Figure 9-9 Thymine dimer,UV induces
a cyclobutane (环丁烷 ) ring between
adjacent T.
Gamma radiation and X-rays
(ionizing radiation) cause double-
strand breaks and are particularly
hazardous (hard to be repaired).
Mutations are also caused by
base analogs and intercalating
agentsDNA
damag
e
?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 mistake during
replication.
?The most mutagenic base anolog
is 5-bromouracil.
?Intercalating agents are flat
molecules containing several
polycyclic rings that interact
with the normal bases in DNA
through hydrogen bonds and
base stacking.
Topic 3,Repair of DNA
damage
CHAPTER 9 The mutability and repair of DNA
3/22/05
Two consequence 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.
Rep
air
of
DNA
damag
e
?Direct reversal of DNA damage by
photoreactivation (光活化作用 ) and
alkyltransferase (烷基转移酶 )
?Base excision repair (切割修复 )
?Nucleotide excision repair
?Recombination (DSB) repairs
?Translesion DNA synthesis
Mechanisms to repair a damage
See Table 9-1 for summary
Rep
air
of
DNA
damag
e
Direct reversal of DNA damage
Error-free repair
Rep
air
of
DNA
damag
e
Photoreactivation
Figure 9-11
Monomerization of thymine dimers
by DNA photolyases in the presence
of visible light.
Methyltransferase
Removes the methyl group from
the methylated O6-methylguanine,
The methyl group is transferred to
the protein itself,inactivating the
protein.
Figure 9-12
Base Excision repair enzyme
remove damaged bases by a base-
flipping mechanismRep
air
of
DNA
damag
e
Glycosylase
? Recognizes the damaged base
? Removes the damaged base
AP endonulease & exonulcease
3.Cleaves the abasic sugars
Exonulcease/DNA
polymerase/ligase
4,Works sequentially to
complete the repair event.
Figure 9-14,
base-flipping
recognition
Figure 9-13,removes the damaged
base and repair
oxoG:A repair,A glycosylase recognizes the
mispair and removes A,A fail-safe
glycosylase also removes T from T:G
mispairs,as if it knows how T is produced?
Fail-safe systems (最后保险系统 )
Figure 9-15:
Nucleotide Excision repair
enzymes cleave damaged DNA on
either side of the lesionRep
air
of
DNA
damag
e
1.Recognize distortions to the
shape of the DNA double helix
2.Remove a short single-
stranded segment that
includes the lesion,
3.DNA polymerase/ligase fill in
the gap.
Figure 9-16**
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
Recombination repairs DNA breaks
by retrieving sequence information
from undamaged DNARep
air
of
DNA
damag
e
Double-strand break (DSB)
repair pathway
Details are in chapter 10
Figure 10-4,Damage
in the DNA template
can lead to DSB
formation during
replication
FIGURE 10-3 DSB repair model for
homologous recombination
Translesion DNA synthesis enables
replication to proceed across DNA
damageRep
air
of
DNA
damag
e
?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,
1.Translesion synthesis is
catalyzed by a specialized
class of DNA polymerases that
synthesize DNA directly
across the damage site,
2.Translesion polymerase is
produced by cell in response
to the DNA damage
3.Translesion polymerases are
expressed as part of the SOS
response pathway,
FIGURE 9-19 Crystal structure of a
translesion polymerase,A Y-family
polymerase found in many organisms,
FIGURE 9-19
Translesion DNA
synthesis in E,coli
重点
3/25/05
掌握 slides里的内容。
CHAPTER 9 The mutability and repair of DNA
Review the lecture
Homework
CHAPTER 9 The mutability and repair of DNA
