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chapter 9 the mutability and repair of dna
Chapter 9 The Mutability and Repair of DNA
  • As we all know that , the perpetuation of the genetic material from generation to generation depends on maintaining rates of mutation at low levels, or the high rates of mutation would destroy the species in the germ line and the individual in the soma.
slide2
At the same time ,if the genetic material were perpetuated with perfect fidelity the genetic variation needed to drive evolution would be lacking ,and new species would not arisen.
  • Therefore ,life and biodiversity depend in a happy balance between mutation and its repair , which is the main content of the chapter 9.
outline
OUTLINE
  • Foreword
  • Replication errors and their repair
  • DNA damage
  • Repair of DNA damage
  • Something unknown
  • Summary
foreword
Foreword

1.Mutation

(1)Definition

Mutations include almost every conceivable change in DNA sequence.

(2)type

point mutation is the mutation that alter a single nucleotide .

a . Transitions (pyrimidine to pyrimidine, purine to purine)

b .Transversions (pyrimidine to purine, purine to pyrimidine

slide5
Insertions

Deletions

Gross 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 )

slide7

3.Two consequences of mutation unrepaired(1)Permanent changes to DNA which can alter the coding sequence of a gene or its regulatory sequence.(2)Some chemical alterations to DNA prevent its use as a template for replication and transcription.So repair of DNA is important to the organisms.OUTLINE

replication errors and their repair
Replication errors and their repair

As we have learned ,the proofreading mechanism ( the 3’→5’ exonuclease component) improves the fidelity of DNA replication, but it is not foolproof.

So there are a total of 12 possible mismatches during the DNA replication (T:T,T:G,T:C, and so forth)

slide9
The misincorporated nucleotide needs to be detected and replaced, otherwise it will cause mutation.
mismatch repair removes errors that escape proofreading
Mismatch repair removes errors that escape proofreading

Now let us discuss the process of repair in detail.

Firstly,E. coli’s repair system

1.How to scan the mismatches and remove errors?

2.How to distinguish the mismatched strand and the parental strand?

1 how to scan the mismatches and remove errors
MutS scans the DNA, recognizing the mismatch from the distortion they cause in the DNA backbone.

MutS is a dimer of the mismatch repair protein.

1.How to scan the mismatches and remove errors?
slide12
MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself.
slide13
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.

MutL is a second protein component of the repair system.

MutH is an enzyme causing an incision or nick on one strand

slide14
The helicase (UrvD) unwinds the DNA starting from the incision and moving in the direction of the site of the mismatch , and the exonucleases progressively digests the displaced single strand , extending to and beyond the site of the mismatched nucleotid.

This action produces a single-stranded gap , which is then filled in by DNA polymerase Ⅲ and sealed with DNA ligase.

2 how to distinguish the mismatched strand and the parental strand
2.How to distinguish the mismatched strand and the parental strand?

The answer is that E. coli tags the

parental strand by transient

hemimethylation as we now

decribe.

slide16
The GATC sequence is widely distributed along the entire genome ,and all of these sites are methylated by the Dam methylase. So before the newly strand is methylated by the Dam methylase after the DNA replication ,the resulting daughter DNA duplexes will be hemimethylated , thus the newly strand is marked (it lacks a methyl group) and hence can be recognized as the strand for repair .
slide17

Different exonucleases are used to remove ssDNA between the nick created by MutH and the mismatch dependoing on whether MutH cuts the DNA in the 5’ or 3’ side of the misoncorporated nucleotide .

secondly the eukaryotic cells repair system
Secondly, the Eukaryotic cells’repair system
  • The Eukaryotic cells repair mismatches and do so using homologs to MutS (MSH) and MutL (MLH).
  • But they lack MutH and E. coli’s clever trick of using heminrthylation to tag the parental strand .
slide19
How does the mismatch repair system of the Eukaryotic cells know which of the two strands to correct ?

As we see in chapter 8, takes place discontinuously wiyh the formation of Okazaki fragments that are joined to previously synthesized DNA by DNA ligase. Prior to the ligation step ,the Okazaki fragment is separated from previously synthesized DNA by a nick created, which can be though of as being equivalent to the nick created in E.coli by MuntH on the newly synthesized strand.OUTLINE

dna 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.

DNA damage
1 hydrolytic damage
The most frequent and important 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.

1.Hydrolytic damage
2 dna damaged by alkylation oxidation and radiation
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.

slide23
Oxidation

DNA is also subject to attack from reactive oxygen species .(eg. O2-, H2O2, and OH•)

■ radiation

ultraviolet light ,which produce the 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
3.caused by base analogs and intercalating agents
  • 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.

slide25
Intercalating agents

They 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 damage
Repair of DNA damage

DNA repair system

  • Direct reversal of DNA damage
  • Excision repair system
  • Recombination (DSB) repairs
  • Translesion DNA synthesis
1 direct reversal of dna damage
1. Direct reversal of DNA damage
  • A repair enzyme simply reverses (undoes) the damage
  • Now we will discuss two examples in detail to understand the direct reversal of DNA damage.
1 photoreactivation
(1).Photoreactivation
  • The enzyme DNA photolyase captures energy from light and it to break the covalent bonds linking adjacent pyrimidines, so the damaged bases are mended directly.
2 the removal of the methyl group
(2).The removal of the methyl group

The methyltransferase

removes the methyl group

from the methylated

O6-methylguanine . The methyl group is transferred to the protein itself, inactivating the protein. (very costly)

2 excision repair system
2. Excision repair system
  • Two kinds of excision repair exist, one involving the removal of only the damaged nucleotide ,and the other ,the removal of a short stretch of single-strand DNA that contains the lesion.
1 base excision repair
(1).Base Excision repair
  • An enzyme called a glycosylase (lesion-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.
what if a damaged base is not removed by base excision before dna replication
What if a damaged base is not removed by base excision before DNA replication ?
  • There are some fail-safe systems to deal with this problem.
  • Then we will discuss two 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 g
T 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 nucleotide excision repair
(2). Nucleotide Excision repair
  • The nucleotide Excision repair enzymes do not recognize any particular lesion, they work by recognizing distortions to the shape of the double helix.
  • Such distortions trigger a chain of events that lead to the removal of short single-stranded segment which is filled in DNA polymerase using the undamaged strand as a template .
the n ucleotide excision repair of e coil
The nucleotide Excision repair of E.coil

(a)UvrA and

UvrB scan

DNA to

identify a

distortion

slide38
(b) UvrA leaves the

complex ,and

UvrB melts DNA

locally around

the distortion

(c)UvrC forms a

complex with UvrB

and creates nicks to

5’ side of the lesion

and to the 3’ side of

the lesion .

(d)DNA helicase UvrD

releases the single

stranded fragment

from the duplex ,and

DNA pol 1and ligase

repair and seal the

gap

transcription coupled repair
Transcription-coupled 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

3 recombination dsb repairs
3. Recombination (DSB) repairs
  • How do cells repair double-strand breaks in DNA in which both strands of the duplex are broken ?
  • 1.When the sister of the broken

chromosome is present in the

cell , the DSB-repair (double

strand break system) pathway

can operate. the DSB-repair

retrieves sequence information

from the sister chromosome. (Details are in chapter 10)

slide41
2.When a chromosome break early in the cell cycle, before a sister has been generated by DNA replication , a fail- safe system comes into play

known as NHEJ

(nonhomologous end joining) .

NHEJ does not involve homologous recombination, instead, the two ends of broken DNA are directly joined to each other by misalignment between single strand protruding from the broken ends.

4 translesion dna synthesis
4. Translesion DNA synthesis
  • If cells cannot repair some lesions, there is a fail-safe mechanism that allows the replication machinery to bypass these sites of damage. This mechanism is known as translesion synthesis.
  • But because of its high error

rate, translesion synthesis can be

considered a system of last resort.

slide43
Translesion synthesis is catalyzed by a specialized class of DNA polymerases that synthesize DNA directly across the site of the damsge.

OUTLINE

something unknown
Something unknown
  • Although the repairs of damaged DNA have formed systems, there are still a lot of problems which have not be solved and need to be studied more deeply.
for example
For example:
  • The mechanism by DNA glycosylase scan for damaged bases remains mysterious
  • In translesion synthesis

1.How does the translesion polymeras recognize a stalled replication fork ?

2.How does the translesion enzyme replace the normal replication polymerase in the DNA replication complex?

3.Once DNA synthesis is extended across the lesion ,how does tge normal replication polymerase switch back to and replace the translesion enzyme at the replication fork? OUTLINE

summary
Summary
  • Now we have learned the whole knowledge on the mutation and repair of DNA ,so we would answer the questions as follows:

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?

slide47
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?

slide48
The main content of this chapter:

we have discussed errors that are generated during replication , lesions that arise from spontaneous damage to DNA , and the damage that is wrought by chemical agents and radiation.

In my opinion , in each case we should consider what cause the alteration to the genetic material , how the alteration to the genetic material is detected and how it is properly repaired. Then if we understand these problems clearly, we must make it!

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