Mutation and Dna Repair Mechanisms

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MUTATIONS AND DNA REPAIR MECHANISMS

MUTATIONS AND DNA REPAIR MECHANISMS



Damage to DNA occurs spontaneously.

Under normal conditions, spontaneoushydrolysis of DNA leads to depurination

(breaking the glycosidic bond betweenthe deoxyribose and the purine) or

deamination (the loss of the amino group

from A, C or G).

This damage can result in the inclusion of an incorrect nucleotide to produce a

mutation.Damage to DNA occurs in response to

mutagens (either chemical or radiation).

Mutagenic chemicals include

1) base analogues (similar in structureto the normal bases and can become

incorporated into DNA);2) base-modifying agents (which can

change a base) and

3) intercalating agents (cause insertions

and deletions).

Ultraviolet (UV) radiation (sunlight) can

cause pyrimidine dimer formation (such

as covalently linked thymines) block



Excision repair mechanisms remove

abnormal nucleotides to correct

mutations.



Base excision repair mechanisms first

remove a damaged base then causesexcision of the remaining sugar-

phosphate unit Pyrimidine dimers and other bulky

lesions are removed through nucleotide

excision repair (NER).

NER causes two nicks which leads tothe removal of a stretch of damaged

single-stranded DNA (12 in E. coli and29 in humans).

Mismatch repair corrects mutations of

non-complementary bases that become

included in DNA during replication that are not fixed by proof-reading.

The original strand is recognized assuch by the action of DNA methylases

(the old DNA strand is methylated).

Mismatch repair endonucleases cut the

sugar phosphate backbone (a nick) andan exonuclease removes the incorrect

nucleotides from the nicked strand.



Mutations can occur in a number of ways:

1.Errors can occur during DNA replication,DNA repair, or DNA recombination which

can lead to base-pair substitutions,

insertions, or deletions, as well as mutations

affecting longer stretches of DNA.

2.Mutagens are chemical or physical agents

that interact with DNA to cause mutations.

3.Physical agents include high-energy

radiation like X-rays and ultraviolet light.

4.Some errors can be corrected by direct

repair, while others are repaired by more

complex mechanisms.



MUTATIONS are changes in the genetic material

of a cell (or virus).

Some are large-scale mutations in which longsegments of DNA are affected (example:

translocations, duplications, and inversions).

A chemical change in just one base pair of a gene

causes a spontaneous or point mutation. A base-pair substitution is a point mutation that

results in replacement of a pair of complimentary

nucleotides with another nucleotide pair.

Some base-pair substitutions have little or noimpact on protein function.

If these occur in gametes or gamete-producing

cells, they may be transmitted to future

generations and cause novel traits or defects.



Transversions (blue): replacement

of a purine by a pyrimidine or that of a pyrimidine by a purine.

Transitions ² (black ): replacement

of one purine by the other or that of

one pyrimidine by the other.

Silent /synonymous mutations changes a codon but

does not alter the amino acid encoded. Alterations of

nucleotides still indicate the same amino acids becauseof redundancy in the genetic code. Such mutations may

still have effects on mRNA stability.

Nonsynonymous mutations result in an altered

sequence in a polypeptide or functional RNA: one or

more components of the sequence are altered oreliminated, or an additional sequence is inserted into

the product.



Missense mutations

are those that still

code for an aminoacid but change the

indicated amino

acid.

Nonsensemutations change

an amino acid

codon into a stop

codon, nearlyalways leading to a

nonfunctional

protein.



Insertions and deletions

are additions or losses of

nucleotide pairs in a gene.

These have a disastrouseffect on the resulting

protein more often than

substitutions do.

Unless these mutations

occur in multiples of three,

they cause a frameshift

mutation.

All the nucleotides

downstream of the deletion

or insertion will be

improperly grouped intocodons.

The result will be extensive

missense, ending sooner or

later in nonsense -

premature termination.



Mutation class Type of mutation Incidence

Base

substitutionsAll types

Comparatively common type of mutation in coding

DNA but also common in noncoding DNA

Transitions and

transversions

Transitions are more common than transversions,

especially in mitochondrial DNA

Synonymous and

nonsynonymous

substitutions

Synonymous substitutions are more common than

nonsynonymous substitutions in coding DNA;

conservative substitutions are more common than

non-conservative

Gene conversion-like

events (multiple base

substitution)

Rare except at certain tandemly repeated loci or

clustered repeats

InsertionsOne or a few

nucleotides

Very common in noncoding DNA but rare in coding

DNA where they produce frameshifts

Triplet repeat

expansions

Rare but can contribute to several disorders,

especially neurological disorders

Other large insertions

Rare; can occasionally get large-scale tandem

duplications, and also insertions of transposable

elements

DeletionsOne or a few

nucleotides

Very common in noncoding DNA but rare in coding

DNA where they produce frameshifts

Larger deletionsRare, but often occur at regions containing tandem

repeats or between interspersed repeats

Chromosomal

abnormalities

Numerical and

structural

Rare as constitutional mutations, but can often be

pathogenic. Much more common as somatic

mutations and often found in tumor cells



1.Purine bases are lost by spontaneous fission of the base-

sugar link.

2.Cytosines, and occasionally adenines, spontaneously

deaminate to produce uracil and hypoxanthine

respectively.

3.Many chemicals, for example alkylating agents, form

adducts with DNA bases.

4.Ultraviolet light causes adjacent thymines to form a

stable chemical dimer.

5.Ionizing radiation causes single or double-strand breaks.

6.Reactive oxygen species in the cell attack purine and

pyrimidine rings.

7.Mistakes in DNA replication result in incorporation of a

mismatched base.

8.Mistakes in replication or recombination leave strand

breaks in DNA.



(A) depurination (loss of purine bases)

resulting from cleavage of the bond between the purine bases and

deoxyribose, leaving an apurinic (AP) site in DNA and (B)

deamination (converts cytosine to uracil; adenine to hypoxanthine)



is the addition of methyl or ethyl groups to

various positions on the DNA bases. Example:

alkylation of guanine by ethylmethane sulfonate

(EMS). At the left is a normal G-C base pair. Note the

free O6 oxygen (red) on the guanine. EMS donates anethyl group (blue) to the O6 oxygen, creating O6-

ethylguanine (right), which base-pairs with thymine

instead of cytosine.



This lesion can be

repaired by an

enzyme (O6-methylguanine

methyltransferase)

that transfers the

methyl group from O6-

methylguanine to acysteine residue in its

active site, and the

original guanine is

restored. This

reaction iswidespread in both

prokaryotes and

eukaryotes, including

humans.



from the sun is carcinogenic

and is a principal cause of skin cancer.

Of the 3 types of ultraviolet radiation (UV) from thesun: UVA (wavelength 320²380 nm), UVB

(wavelength 290²320 nm), and UVC (wavelength

200²290 nm, UVB is the most effective carcinogen

because it causes UV photoproducts. Cyclobutane pyrimidine dimers are responsible for at

least 80% of UVB-induced mutations. The precise

class of mutations resulting from pyrimidine dimers

is a unique molecular signature of skin cancer.

UVA indirectly damages DNA via free radical-mediated damage. Water is fragmented by UVA,

generating electron-seeking ROS that cause DNA

damage (transversions are characteristic of UVA

damage).



most common type of DNA damage

caused by UV irradiation. (a) UV light cross-links the

two thymine bases on the top strand. This distorts the

DNA so that these two bases no longer pair with their

adenine partners. (b) The two bonds joining the two

thymines form a 4-membered cyclobutane ring (red).



17

UV-induced thymine dimers can

be repaired by

photoreactivation. The enzyme

(photolyase) absorbs visible

light and binds to damaged

DNA. The enzyme breaks the

dimer, and finally dissociates

from the repaired DNA. Repair

of pyrimidine dimers by

photoreactivation is common to

prokaryotic and eukaryotic

cells, including E. coli, yeasts,

and some species of plants and

animals. Photoreactivation is

not universal; many species

(including humans) lack this

mechanism of DNA repair.



high-energy radiation capable of

producing ionization in substances through which it

passes, e.g. x-rays, alpha and beta rays, and neutrons froma nuclear reaction.

It can directly ionize atoms comprising DNA, or

indirectly by the interaction with water molecules

(radiolysis) that generate dangerous reactive oxygen

species (ROS): the hydroxyl radical (²OH), hydrogenperoxide (H2O2), and the superoxide radical (O ²

2).

A free radical reacts very strongly with other molecules

as it seeks to restore a stable configuration of electrons.

A free radical may drift about up to 1010 longer than the

time needed for the initial ionization, increasing thechance of it disrupting DNA and cause mutations.



Oxidation of DNA is one of the main causes of

mutation, and explains why free radicals are such

potent carcinogens. Oxidation can produce oxidized bases, e.g., adenine

mispairs with 8-oxoguanine during replication

leading to a GT transversion mutation.

The -OH radical removes electrons from any

molecule in its path, turning that molecule into a free

radical and so propagating a chain reaction.

H2O2 is more dangerous to DNA than the -OH

radical. Its slower reactivity gives it time to travel

into the nucleus of a cell, where it is free to wreakhavoc upon DNA.

The superoxide radical is not very reactive but acts

more as a catalyst for the generation of the other

ROS intermediates.



:

The common mechanism of action is that an electrophilic

(electron-deficient) form reacts with nucleophilic sites (sites

that can donate electrons) in the purine and pyrimidine ringsof nucleic acids.

Some chemicals are base analogues that may be substituted

into DNA, and pairs incorrectly during DNA replication.

Other mutagens interfere with DNA replication by inserting

into DNA and distorting the double helix. Still others cause chemical changes in bases (DNA adducts)

that change their pairing properties.

Carcinogens can be segregated into 10 groups:

polycyclic aromatic hydrocarbons carbamates

halogenated compounds aromatic aminesnitrosamines and nitrosamides azo dyes

hyrazo and azoxy compounds natural products

inorganic carcinogens

miscellaneous compounds (alkylating agents,

aldehydes, phenolics)



) are

carcinogens produced by cooking

meat, formed from heating amino acids

and proteins. About 20 HCAs havebeen identified. Three examples, Phe-

P-1, IQ, and Mel Q, are shown.

These are examples of carcinogens to

which we are exposed daily and whichare produced in our own kitchens!

Oven roasting, marinading, and coatingfood with breadcrumbs before fryingare modifications that may reduce the

formation of HCAs.



(a) An example of nitrosamines: alkylnitrosoureas.

(b) A potential carcinogenic product of nitrosamines: O6 adduct of

guanine. Guanine is shown for comparison.

are found in tobacco or are

formed when preservative

nitrites react with amines in fishand meats during smoking. Their

principal carcinogenic product is

alkylated O6 guanine derivatives.



treatment of DNA results in the

conversion of adenine into hypoxanthine, which pairs

with cytosine, inducing a transition from A-T to G-C.

induce frameshift mutations by

intercalating into the DNA, leading to the incorporation

of an additional base on the opposite strand.



. The compound, produced by moldsthat grow on peanuts, is activated by cytochrome

P450 to form a highly reactive species that modifies

bases such as guanine in DNA, leading to mutations.



The bases of DNA can exist in rare

tautomeric forms. The imino

tautomer of adenine can pair with

cytosine, eventually leading to atransition from A-T to G-C.

(Tautomerization is the

interconversion of two isomers that

differ only in the position of protons

and often, double bonds).

This base

analog of thymine has a

higher tendency to form an

enol tautomer than does

thymine itself. The pairing of

the enol tautomer of 5-

bromouracil with guanine will

lead to a transition from T-A

to C-G.



(a)(b) Metabolic activation of BP (Benzopyrene)(a)(b) Metabolic activation of BP (Benzopyrene)



Benzopyrene ( found in cigarette smoke) reacts with DNA

bases, resulting in the addition of large bulky chemical groups

to the DNA molecule. Locations of these adducts matched the

distribution of p53 gene mutations in lung tumors from

smokers (Science,1996).

Each day the DNA of a human cell loses about 5,000 purines,

and about 100 cytosines spontaneously deaminate to uracil.

Damage to DNA can block replication or transcription, and can

result in a high frequency of mutations³consequences that are

unacceptable from the standpoint of cell reproduction.



Major DNA repairing mechanisms: base excision,nucleotide excision and mismatch repair.



A DNA glycosylase specific for G-T

mismatches, usually formed by

deamination of 5-methyl C residues, flipsthe thymine base out of the helix and then

cuts it away from the sugar-phosphate

DNA backbone (1), leaving just the

deoxyribose (black dot). An endonuclease

specific for the resultant baseless site thencuts the DNA backbone (2), and the

deoxyribose phosphate is removed by an

endonuclease associated with DNA

polymerase (3). The gap is then filled in by

DNA Pol and sealed by DNA ligase (4),

restoring the original G-C base pair.



DNA's bases may bemodified by deamination

or alkylation. The position

of the modified

(damaged) base is called

the "abasic site" or "AP

site". DNA glycosylase

can recognize the AP site

and remove its

base. Then, the AP

endonuclease removes

the AP site andneighboring

nucleotides. The gap is

filled by DNA polymerase I

and DNA ligase.



Proteins UvrA,

UvrB, and UvrC are

involved in removing the

damaged nucleotides

(e.g., the dimer inducedby UV light). The gap is

then filled by DNA

polymerase I and DNA

ligase. In yeast, the

proteins similar to Uvr'sare named RADxx

(radiation), such as

RAD3, RAD10, etc.



A DNA lesion that

causes distortion of the double

helix, such as a thymine dimer, isinitially recognized by a complex

of the XP-C (Xeroderma

pigmentosum C protein) and 23B

proteins (1). This complex then

recruits transcription factor TFIIH,

whose helicase subunits, poweredby ATP hydrolysis, partially unwind

the double helix. XP-G and RPA

proteins then bind to the complex

and further unwind and stabilize

the helix until a bubble of §25

bases is formed (2). Then XP-G

(now acting as an endonuclease)

and XP-F, a 2nd endonuclease, cut

the damaged strand at points 24²

32 bases apart on each side of the

lesion (3).



This releases the DNA fragment with the damaged

bases, which is degraded to mononucleotides.

Finally the gap is filled by DNA polymerase exactly as

in DNA replication, and the remaining nick is sealed by

DNA ligase (4 )



The mismatch repair system

detects and excises

mismatched bases in newlyreplicated DNA, which is

distinguished from the

parental strand because it has

not yet been methylated. MutS

binds to the mismatched base,

followed by MutL. The bindingof MutL activates MutH, which

cleaves the unmodified strand

opposite a site of methylation.

MutS and MutL, together with

helicase II, SSB proteins, and

an exonuclease, then excisethe portion of the unmodified

strand that contains the

mismatch. The gap is then

filled by DNA polymerase and

sealed by ligase.



Mismatch repair in eukaryotes may be

similar to that in E. coli. Homologs of MutS

and MutL have been identified in yeast,

mammals, and other eukaryotes. MSH1 to

MSH5 are homologous to MutS; MLH1,PMS1 and PMS2 are homologous to MutL.

Mutations of MSH2, PMS1 and PMS2 are

related to colon cancer.

In eukaryotes, the mechanism to distinguishthe template strand from the new strand is

still unclear.



A complex of the

MSH2 and MSH6 proteins bindsto a mispaired segment of DNA

such as to distinguish between

the template and newly

synthesized daughter strands

(1). This triggers binding of the

MLH1 endonuclease, as well as

other proteins such as PMS2,which has been implicated in

onco-genesis through mismatch-

repair mutations. A DNA helicase

unwinds the helix and the

daughter strand is cut; an

exonuclease then removesseveral nucleotides, including

the mismatched base (2). Finally,

as with base excision repair, the

gap is then filled in by a DNA

polymerase (Pol, in this case)

and sealed by DNA ligase (3 ).



The presence of a thymine

dimer blocks replication, but

DNA polymerase can bypass

the lesion and reinitiatereplication at a new site

downstream of the dimer. The

result is a gap opposite the

dimer in the newly synthesized

DNA strand. In

recombinational repair, thisgap is filled by recombination

with the undamaged parental

strand. Although this leaves a

gap in the previously intact

parental strand, the gap can

be filled by the actions of

polymerase and ligase, usingthe intact daughter strand as a

template. Two intact DNA

molecules are thus formed,

and the remaining thymine

dimer eventually can be

removed by excision repair.



If the replication fork encounters an

unrepaired lesion or strand break, replication generally halts and the fork may

collapse. A lesion is left behind in an unreplicated, single-stranded segment of

the DNA; a strand break becomes a double-strand break.

There are two possible

avenues for repair:

recombinational DNA

repair or, when lesions

are unusually

numerous, error-prone

repair. The latter

involves DNA

polymerase V,

encoded by the umuC

and umuD genes that

can inaccurately

replicate over manytypes of lesions. The

repair mechanism is

referred to as error-

prone because

mutations often result.



UV light activates the

RecA co-protease,which stimulates the

LexA protein (purple)

to cleave itself,

releasing it from the

umuDC operon. This

results in synthesisof UmuC and UmuD

proteins, which

somehow allow DNA

synthesis across

from a thymine dimer,

even though mistakes(blue) will be made.



The black and

red DNAs represent thehomologous sequences on

sister chromatids. (1) A double-

strand DNA break forms in thechromatids. (2) The double-

strand break activates the ATM

kinase; this leads to activationof a set of exonucleases that

remove nucleotides at the break

from the 3· and 5· ends of both

broken strands, ultimately

creating single stranded 3· ends.In a process that is dependent

on the BRCA1 and BRCA2

proteins, as well as others, the

Rad51 protein (green ovals)

polymerizes on single-stranded

DNA with a free 3· end to form anucleoprotein filament.



(3): Aided by yet other

proteins, one Rad51

nucleoprotein filament

searches for thehomologous duplex DNA

sequence on the sisterchromatid, then invades

the duplex to form a joint

molecule in which the

single stranded 3· end isbase-paired to the

complementary strand onthe homologous DNA

strand. (4) The replicative

DNA polymerases

elongate this 3· end of the

damaged DNA (green

strand), templated by thecomplementary

sequences in theundamaged homologous

DNA segment.



(5) Next this repaired

3· end of the damaged

DNA pairs with the

single stranded 3· endof the other damaged

strand. (6) Any

remaining gaps are

filled in by DNA

polymerase and ligase(light green),

regenerating a wild-

type double helix in

which an entire

segment (dark andlight green) has been

regenerated from the

homologous segment

of the sister

chromatid.



In general, nucleotide sequences

are butted together that were not

apposed in the unbroken DNA.

These DNA ends are usually from

the same chromosome locus, and

when linked together, several base

pairs are lost. Occasionally, ends

from different chromosomes are

accidentally joined together. Acomplex of two proteins, Ku and

DNA-dependent protein kinase,

binds to the ends of a double-strand

break (1). After formation of a

synapse, the ends are further

processed by nucleases, resulting

in removal of a few bases (2), and

the two double-stranded molecules

are ligated together (3). As a result,

the double-strand break is repaired,

but several base pairs at the site of

the break are removed.



If DNA can repair itself,

Go ahead, indulge yourself andenjoy life·s pleasures!

After all, life is short «

But DNA can only do so much foritself«

Abusing its potentials can cause YOUand your future generations

major, major problems!

Mutation and Dna Repair Mechanisms

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