Chapter 9 The Mutability Chapter 9 The Mutability and Repair of DNA and Repair of DNA As we all know that , the As we all know that , the perpetuation of the genetic perpetuation of the genetic material from generation to material from generation to generation depends on generation depends on maintaining rates of maintaining rates of mutation at low levels, or mutation at low levels, or the high rates of mutation the high rates of mutation would destroy the species would destroy the species in the germ line and the in the germ line and the individual in the soma. individual in the soma.
Transcript
Chapter 9 The Mutability Chapter 9 The Mutability and Repair of DNAand Repair of DNA As we all know that , the As we all know that , the
perpetuation of the genetic perpetuation of the genetic material from generation to material from generation to generation depends on generation depends on maintaining rates of mutation at maintaining rates of mutation at low levels, or the high rates of low levels, or the high rates of mutation would destroy the mutation would destroy the species in the germ line and the species in the germ line and the individual in the soma.individual in the soma.
At the same time ,if the genetic At the same time ,if the genetic material were perpetuated with material were perpetuated with perfect fidelity the genetic perfect fidelity the genetic variation needed to drive variation needed to drive evolution would be lacking ,and evolution would be lacking ,and new species would not arisen.new species would not arisen.
Therefore ,life and biodiversity Therefore ,life and biodiversity depend in a happy balance depend in a happy balance between mutation and its between mutation and its repair , which is the main repair , which is the main content of the chapter 9.content of the chapter 9.
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ForewordForeword Replication errors and their repair Replication errors and their repair DNA damage DNA damage Repair of DNA damageRepair of DNA damage Something unknown Something unknown Summary Summary
ForewordForeword1.Mutation 1.Mutation (1)Definition (1)Definition Mutations include almost every Mutations include almost every
conceivable change in DNA sequence.conceivable change in DNA sequence. (2)type(2)type point mutationpoint mutation is the mutation that alter a is the mutation that alter a
single nucleotide .single nucleotide . a . a . Transitions (pyrimidine to pyrimidine,
purine to purine) b .Transversions (pyrimidine to purine,
purine to pyrimidine
InsertionsDeletionsGross rearrangement of
chromosome “hotspots”: the sites on the
chromosome where the mutations arise at high frequency. (eg. DNA microsatellite ,Mutation-prone sequence in human genome are repeats of simple di-, tri- or tetranucleotide sequences, these sequences (1) are important in human genetics and disease, (2) hard to be copied accurately and highly polymorphic in the population )
2.Three important sources for mutation 2.Three important sources for mutation (unavoidable)(unavoidable)
Three important sources
for mutation
Inaccuracy in DNA replication
Chemical damage to the genetic material
The insertions generated by DNA elements
known as transposition
(Consider in detail in Chapter 11)
3.Two consequences of mutation 3.Two consequences of mutation unrepairedunrepaired((1)Permanent changes to DNA 1)Permanent changes to DNA which can alter the coding which can alter the coding sequence of a gene or its regulatory sequence of a gene or its regulatory sequence.sequence.(2)Some chemical alterations to DNA (2)Some chemical alterations to DNA prevent its use as a template for prevent its use as a template for replication and transcription.replication and transcription. So repair of DNA is important to the So repair of DNA is important to the organisms.organisms. OUTLINEOUTLINE
Replication errors and Replication errors and their repairtheir repairAs we have learned ,the As we have learned ,the
proofreading mechanism ( the proofreading mechanism ( the 3’→5’ exonuclease 3’→5’ exonuclease component) improves the fidelity component) improves the fidelity of DNA replication, but it is not of DNA replication, but it is not foolproof.foolproof.
So there are a total of 12 possible So there are a total of 12 possible mismatches during the DNA mismatches during the DNA replication (T:T,T:G,T:C, and so replication (T:T,T:G,T:C, and so forth)forth)
The misincorporated nucleotide needs to be detected and replaced, otherwise it will cause mutation.
Mismatch repair removes errors that escape proofreading
Now let us discuss the process of Now let us discuss the process of repair in detail.repair in detail.
Firstly,Firstly, E. coli’s repair system
1.How to scan the mismatches 1.How to scan the mismatches and remove errors?and remove errors?
2.How to distinguish the mismatc2.How to distinguish the mismatched strand and the parental strahed strand and the parental strand?nd?
1.How to scan the mismatches 1.How to scan the mismatches and remove errors?and remove errors?
MutSMutS scans the DN scans the DNA, recognizing thA, recognizing the mismatch from e mismatch from the distortion thethe distortion they cause in the DNy cause in the DNA backbone.A backbone.
MutS is a dimer of tMutS is a dimer of the mismatch repahe mismatch repair protein.ir protein.
MutS embraces the misMutS embraces the mismatch-containing DNmatch-containing DNA, inducing a pronouA, inducing a pronounced kink in the DNA nced kink in the DNA and a conformationaand a conformational change in MutS itsel change in MutS itself.lf.
MutS-mismatch-containinMutS-mismatch-containing DNA complex recruitg DNA complex recruits s MutL,MutL, MutL activates MutL activates MutH,MutH, an enzyme causi an enzyme causing an incision or nick ong an incision or nick on one strand near the sin one strand near the site of the mismatch.te of the mismatch.
MutL is a second protein MutL is a second protein component of the repaicomponent of the repair system.r system.
MutH is an enzyme causinMutH is an enzyme causing an incision or nick on g an incision or nick on one strand one strand
The helicase (UrvD) unwiThe helicase (UrvD) unwinds the DNA starting fronds the DNA starting from the incision and movinm the incision and moving in the direction of the sig in the direction of the site of the mismatch , and tte of the mismatch , and the exonucleases progreshe exonucleases progressively digests the displacsively digests the displaced single strand , extendied single strand , extending to and beyond the sitng to and beyond the site of the mismatched nucle of the mismatched nucleotid.eotid.
This action produces a siThis action produces a single-stranded gap , whicngle-stranded gap , which is then filled in by DNA h is then filled in by DNA polymerase and sealeⅢpolymerase and sealeⅢd with DNA ligase. d with DNA ligase.
2.How to distinguish the 2.How to distinguish the mismatched strand and the mismatched strand and the parental strand?parental strand?
The answer is that The answer is that E. coli tags the tags the
parental strand by transientparental strand by transient
hemimethylation as we nowhemimethylation as we now
decribe.decribe.
The GATC sequence is widely distributThe GATC sequence is widely distributed along the entire genome ,and all of ted along the entire genome ,and all of these sites are methylated by the Dam hese sites are methylated by the Dam methylase. So before the newly strand imethylase. So before the newly strand is methylated by the Dam methylase aftes methylated by the Dam methylase after the DNA replication ,the resulting daur the DNA replication ,the resulting daughter DNA duplexes will be hemimethylghter DNA duplexes will be hemimethylated , thus the newly strand is marked (iated , thus the newly strand is marked (it lacks a methyl group) and hence can bt lacks a methyl group) and hence can be recognized as the strand for repaire recognized as the strand for repair . .
Different exonucleases are used to rDifferent exonucleases are used to remove ssDNA between the nick creaemove ssDNA between the nick created by MutH and the mismatch depeted by MutH and the mismatch dependoing on whether MutH cuts the Dndoing on whether MutH cuts the DNA in the 5’ or 3’ side of the misoncNA in the 5’ or 3’ side of the misoncorporated nucleotideorporated nucleotide . .
SecondlySecondly, , the the Eukaryotic cells’ Eukaryotic cells’ repair system
The The Eukaryotic cells repair miEukaryotic cells repair mismatches and do so using hosmatches and do so using homologs to MutS (MSH) and Mumologs to MutS (MSH) and MutL (MLH). tL (MLH).
But they lack MutH and But they lack MutH and E. coli’s clever trick of using heminrthylation to tag the parental strand .
How does the mismatch repair How does the mismatch repair system of the Eukaryotic cells system of the Eukaryotic cells know which of the two strands to know which of the two strands to correct ?correct ? As we see in chapter 8, takes place diAs we see in chapter 8, takes place di
scontinuously wiyh the formation of scontinuously wiyh the formation of Okazaki fragments that are joined to Okazaki fragments that are joined to previously synthesized DNA by DNA previously synthesized DNA by DNA ligase. Prior to the ligation step ,the ligase. Prior to the ligation step ,the Okazaki fragment is separated from Okazaki fragment is separated from previously synthesized DNA by a nicpreviously synthesized DNA by a nick created, which can be though of as k created, which can be though of as being equivalent to the nick created ibeing equivalent to the nick created in E.coli by MuntH on the newly synthn E.coli by MuntH on the newly synthesized strand.esized strand.OUTLINEOUTLINE
DNA damageDNA damage
DNA undergoes damage spontaneously from hydrolysis and deamination.
DNA damaged by alkylation , oxidation and radiation.
Mutations are also caused by base analogs and intercalating agents.
1.Hydrolytic damage
The most frequent and iThe most frequent and important kind of hmportant kind of hydrolytic damage is deamination of the base cytosine, (just show in the picture on the left)
Notice that , in contrast to the replication errors ,all of these hydrolytic reactions result in alterations to the DNA that are unnatural.
2.DNA damaged by alkylation , oxidation and radiation Alkylation
In alkylation ,methyl
or ethyl groups are transferred to reactive
sites on the baese and to phosphates in the DNA backcone.
Oxidation DNA is also subject to attack
from reactive oxygen species .(eg. O2-, H2O2, and OH•)
■ radiation ultraviolet light ,which produce t
he photochemical fusion of two pyrimidines that occupy adjacent positions on the same polynucleotide chain. (eg. thymine dimer) These linked bases are incapable of base-pairing and cause the DNA polymerase to stop during replication.
gamma radiation and X-ray, which cause double-strand breaks in the DNA ,which are different to repair.
3.caused by base analogs and intercalating agents Base analogsSimilar 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.
Intercalating agentsThey are flat molecules containing
several polycyclic rings that rings that bind to the equally flat purine or pyrimidine bases of DNA, just as the bases bind or stack with each other in the double helix. OUTLINE
Repair of DNA damageRepair of DNA damage
DNA repair system DNA repair system Direct reversal of DNA damage Excision repair system Recombination (DSB) repairs Translesion DNA synthesis
1. 1. Direct reversal of DNA damage
A repair enzyme simply reverseA repair enzyme simply reverses (undoes) the damages (undoes) the damage
Now we will discuss two examplNow we will discuss two examples in detail to understand the es in detail to understand the direct reversal of DNA damage.
(1).Photoreactivation(1).Photoreactivation
The enzyme DNA photolyase cThe enzyme DNA photolyase captures energy from light and it taptures energy from light and it to break the covalent bonds linkio break the covalent bonds linking adjacent pyrimidines, so the ng adjacent pyrimidines, so the damaged bases are mended dirdamaged bases are mended directly.ectly.
(2).The removal of the methyl (2).The removal of the methyl groupgroupThe methyltransferase removes the methyl group from the methylated O6-methylguanine . The methyl gr
oup is transferred to the protein itself, inactivating the protein. (very costly)
2. 2. Excision repair system
Two kinds of excision repair Two kinds of excision repair exist, one involving the removal exist, one involving the removal of only the damaged of only the damaged nucleotide ,and the other ,the nucleotide ,and the other ,the removal of a short stretch of removal of a short stretch of single-strand DNA that contains single-strand DNA that contains the lesion. the lesion.
(1).Base Excision repair An enzyme called a gAn enzyme called a glycosylase (le
sion-specific) recognizes and removes the damaged base by hydrolyzing the glycosidic bond.
The resulting abasic sugar is removed from the DNA backbone.
After the damaged nucleotide has been entirely removed from the backbone, a repair DNA polymerase and DNA ligase restore an intact strand using the undamaged strand as a template.
removes the damaged base and repair
What if a damaged base is not What if a damaged base is not removed by base excision before removed by base excision before DNA replication ? DNA replication ?
There are some fail-safe There are some fail-safe systems to deal with this systems to deal with this problem.problem.
Then we will discuss two Then we will discuss two examples in detail.examples in detail.
oxoG mispair with A oxoG mispair with A
A dedicated glycosylase which recognizes the oxoG:A base pairs recognizes an A opposite an oxoG as a mutation and removed the undamaged but incorrect base.
T mispair with GT mispair with G
A glycosylase removes T from T:G mispairs.
The glycosylase system assumes that the T in the T:G mismatch arose from deamination of 5-methyl-cytosine and selectively removes the T so that it can be replaced with a C.
(2). (2). Nucleotide Excision repair The nThe nucleotide Excision repair
enzymes do not recognize any enzymes do not recognize any particular lesion, they work by particular lesion, they work by recognizing distortions to the shape recognizing distortions to the shape of the double helix.of the double helix.
Such distortions trigger a chain of Such distortions trigger a chain of events that lead to the removal of events that lead to the removal of short single-stranded segment which short single-stranded segment which is filled in DNA polymerase using the is filled in DNA polymerase using the undamaged strand as a template . undamaged strand as a template .
The nThe nucleotide Excision repair of E.coil E.coil
(a)UvrA and (a)UvrA and
UvrB scan UvrB scan
DNA to DNA to
identify a identify a
distortion distortion
(b) UvrA leaves the (b) UvrA leaves the complex ,and complex ,and UvrB melts DNA UvrB melts DNA locally around locally around the distortionthe distortion
(c)UvrC forms a (c)UvrC forms a complex with UvrB complex with UvrB and creates nicks to and creates nicks to 5’ side of the lesion 5’ side of the lesion
and to the 3’ side of and to the 3’ side of
the lesion .the lesion .
(d)DNA helicase UvrD (d)DNA helicase UvrD releases the single releases the single stranded fragment stranded fragment from the duplex ,and from the duplex ,and DNA pol 1and ligase DNA pol 1and ligase repair and seal the repair and seal the gapgap
excision repair (NER) system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template
3. 3. Recombination (DSB) repairs How do cells repair double-strand How do cells repair double-strand
breaks in DNA in which both strands breaks in DNA in which both strands of the duplex are broken ?of the duplex are broken ?
1.When the sister of the broken 1.When the sister of the broken chromosome is present in the chromosome is present in the cell , the DSB-repair (double cell , the DSB-repair (double strand break system) pathway strand break system) pathway can operate. the DSB-repair can operate. the DSB-repair retrieves sequence information retrieves sequence information from the sister chromosome. from the sister chromosome.
((Details are in chapter 10)
2.When a chromosome break early i2.When a chromosome break early in the cell cycle, before a sister has bn the cell cycle, before a sister has been generated by DNA replication , een generated by DNA replication , a fail- safe system comes into play a fail- safe system comes into play
known as NHEJ known as NHEJ (nonhomologous end joining) .(nonhomologous end joining) . NHEJ does not involve homologous NHEJ does not involve homologous
recombination, instead, the two ends recombination, instead, the two ends of broken DNA are directly joined to of broken DNA are directly joined to each other by misalignment between each other by misalignment between single strand protruding from the brosingle strand protruding from the broken ends. ken ends.
4. 4. Translesion DNA synthesis If cells cannot repair some lesions, thIf cells cannot repair some lesions, th
ere is a fail-safe mechanism that alloere is a fail-safe mechanism that allows the replication machinery to bypaws the replication machinery to bypass these sites of damage. This mechss these sites of damage. This mechanism is known as translesion synthanism is known as translesion synthesis. esis.
But because of its high error But because of its high error
rate, translesion synthesis can be rate, translesion synthesis can be
considered a system of last resort. considered a system of last resort.
TranslesiTranslesion synthesis on synthesis is catalyzed is catalyzed by a specializby a specialized class of Ded class of DNA polymeraNA polymerases that syntses that synthesize DNA hesize DNA directly acrosdirectly across the site of ts the site of the damsge.he damsge.
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Something unknown Something unknown
Although the repairs of damageAlthough the repairs of damaged DNA have formed systems, thd DNA have formed systems, there are still a lot of problems whiere are still a lot of problems which have not be solved and need ch have not be solved and need to be studied more deeply. to be studied more deeply.
For example:For example: The mechanism by DNA gThe mechanism by DNA glycosylase
scan for damaged bases remains mysterious
In translesion synthesistranslesion synthesis 1.How does the translesion polymeras r1.How does the translesion polymeras r
ecognize a stalled replication fork ?ecognize a stalled replication fork ? 2.How does the translesion enzyme repl2.How does the translesion enzyme repl
ace the normal replication polymerase iace the normal replication polymerase in the DNA replication complex?n the DNA replication complex?
3.Once DNA synthesis is extended acro3.Once DNA synthesis is extended across the lesion ,how does tge normal repliss the lesion ,how does tge normal replication polymerase switch back to and recation polymerase switch back to and replace the translesion enzyme at the repliplace the translesion enzyme at the replication fork? cation fork? OUTLINEOUTLINE
Summary Summary
Now we have learned the whole Now we have learned the whole knowledge on the mutation and knowledge on the mutation and repair of DNA ,so we would repair of DNA ,so we would answer the questions as followsanswer the questions as follows::
1.How is the DNA mended 1.How is the DNA mended rapidly enough to prevent rapidly enough to prevent errors from becoming set in the errors from becoming set in the genetic material as mutation? genetic material as mutation? 2.How does the cell distinguish the 2.How does the cell distinguish the parental strand from the daughter parental strand from the daughter strand in repairing replication errors?strand in repairing replication errors?
3.How does the cell restore 3.How does the cell restore
the proper DNA sequence the proper DNA sequence
when the original sequencewhen the original sequence
can no longer be read?can no longer be read?
4.How does the cell deal with 4.How does the cell deal with
lesions that block replication?lesions that block replication?
The main content of this chapter:The main content of this chapter: we have discussed errors that are we have discussed errors that are
generated during replication , lesions generated during replication , lesions that arise from spontaneous damage that arise from spontaneous damage to DNA , and the damage that is to DNA , and the damage that is wrought by chemical agents and wrought by chemical agents and radiation.radiation.
In my opinion , in each case we In my opinion , in each case we should consider what cause the should consider what cause the alteration to the genetic material , alteration to the genetic material , how the alteration to the genetic how the alteration to the genetic material is detected and how it is material is detected and how it is properly repaired. Then if we properly repaired. Then if we understand these problems clearly, understand these problems clearly, we must make it! we must make it!
