dr. asad vaisi-rayggani department of clinical biochemistry kermanshah university of medical...

Dr. Asad Vaisi-Rayggani

Department of Clinical Biochemistry

Kermanshah University of Medical Sciences

DNA Repair and

Mutation• Definition: • Mutations are inheritable changes in the DNA sequence.

They can result from replication errors, from damage to the DNA, or from errors introduced during repair of damage. Mutations that are changes of a single base pair are called point mutations.

• Causes:

It may be spontaneous or induced because of different agents

• Classifications:

are classified on different basis

Enzymes Repair Damaged DNA• A human has 1014 nucleated cells each with 3x

109 base pairs of DNA. If about 1016 cell divisions occur in a lifetime and

• 10−10 mutations per base pair per cell generation escape repair,

• there may eventually be as many as one mutation per 106 bp in the genome.

• Fortunately,most of these will probably occur in DNA that does not encode proteins or will not affect the function of encoded proteins and so are of no consequence.

• In addition,spontaneous and chemically induced damage to DNA must be repaired.

• Damage to DNA by environmental, physical, and chemical agents may be classified into four types

• (Table 36–8).

Different Causes of mutations: Contrary to popular belief…Most DNA damage is caused by endogenous mutagens

Estimated DNA damage/day in human cells

SSBs ~50,000/day Depurinations ~10,000/day Deaminations ~600/day Oxidations ~2000/day Alkylations ~5000/day DSBs ~50-100/day -

PHYSICAL MUTAGENS / RADIATION

• radiation was discovered in the 1890s -Roentgen discovered X-rays in 1895 -Becquerel discovered radiation in 1896 -Marie and Pere Curie discovered radioactive elements in 1898• first discovered mutagenic agent known -effects on genes first reported in 1920s in Drosophila (Muller)

BIOLOGICALLY SIGNIFICANT

EM spectrum -consists of electric

and magnetic waves

• 1- Base analogs: resemble purines and pyrimidines – bromouracil (BU) & aminopurine

CHEMICAL MUTAGENS

CHEMICAL MUTAGENS

They are:• Flat, multiple ring molecules, that can interact with and insert between DNA bases.

• It Causes:• DNA to be stretched• Insertinon of an extra base opposite intercalated molecule by DNA polymerase = FRAMESHIFT MUTATION

acridine orange ethidium bromide proflavin

2- intercalating agents

3- Nitrous acid:

cause deaminations C U, meC T

A hypoxanthine

4-Nitrosoguanidine cause base alkylation

methyl and ethyl

methanesulfonate

5-Hydroxylamine Hydroxylates amino-gp of

C pairs with A

CHEMICAL MUTAGENS

1- Sterilization: Induction of mutation

to sterile germs. .

Mutagenesis as a tool !

a: Oxidation: It is caused by: 1- Normal metabolism 2- ROS (reactive oxygen species) such as O2

-, H2O2, OH.

3- Ionizing radiation 4- Chemicals It causes: Base-mispairing (i.e., oxoG can pair with C or A)

Base alteration/damage

Base alteration/damageb: Alkylation:

It is caused by: Transfer of methyl or ethyl group to DNA bases

It causes Base-mispairing (ie., O6-methylG mispairs with T)

• Transitions are point mutations in which one purine is substituted for another (i.e., A for G or G for A) or

• One pyrimidine is substituted for another( i.e.,T for C or C for T). Deamination of C to form U,

Point mutations can also be characterized by their effect on a coding sequence

• Missense mutations are point mutations that change a single base pair in a codon such that the codon now encodes a different amino acid

• Nonsense mutations are point mutations that change a single base pair in a codon to a stop codon that terminates translation

• (Figure4 .24b)

• Nonsense mutations usually have more severe effects than missense mutations, because they lead to synthesis of truncated (and generally unstable) polypeptides

• Silent or synonymous mutations do not alter the amino acid encoded; these include

• many changes in the third nucleotide of a codon. Some silent mutations may, however,

• Have serious consequenceis they alter the splicing pattern of the gene.

• Insertions or deletions of one or more base pairs( if the number of base pairs is not a multiple of 3) lead to frame shift that disrupt the coding of a protein

• acridines and proflavin, intercalate into the

• DNA; that is, they insert between adjacent base pairs. This usually leads to insertions or deletions of a single base pair, and thus a frame shift

Bruce Nathan AmesBruce Nathan AmesBrith:1928Brith:1928Ames test: 1970Ames test: 1970

Can we detect Mutagen: Ames Assay

For carcinogens, based on their mutagencitySalmonula having a mutant that in active an enzyme of the His biosynthetic90% found in eukaryotic

DNA Damage, Repair, and Consequences

Damaging agent

Consequences

Repair Process

In hibition of:

•Replication

•Transcription

•Chromosome segregation•Mutation

•Chromosome aberration

DNA Repair Pathways

4. Recombinational repair - multiple pathways - double strand breaks and inter strand cross-links

1. Direct reversals2. Excision repair

a. Base excision repair (BER) b. Nucleotide excision repair (NER)

3. Mismatch repair - replication errors

4. Recombinational repair - multiple pathways - double strand breaks and inter strand cross-links

5. Tolerance mechanisms

Damage Recognized:Thymine dimers6-4 photoproduct

Gene Products Required:Photolyase

Related disease:Photolyase not yet found in placental mammals

T T

Visible light

T T

1- Direct reversal: photoreactivation

Mismatch Repair

• Faulty(Damaged) mismatch repair has been linked to hereditary nonpolyposis colon cancer (HNPCC), one of the most common inherited cancers.

• Genetic studies linked HNPCC in some families to a region of chromosome 2.

• The gene located, designated hMSH2, was subsequently shown to encode the human analog of the E coli MutS protein

• Mutations of hMSH2 account for 50–60% of HNPCC cases.

Base Excision-Repair• The depurination of DNA, which happens

spontaneously owing to the thermal lability of the purine Nglycosidic bond, occurs at a rate of 5000–10,000/cell/d at 37 °C.

• Specific enzymes recognize a depurinated site and replace the appropriate purine directly, without interruption of the phosphodiester backbone.

Uracil

apurinic or apyrimidinic endonucleaseto excise the abasic sugar.

Nucleotide Excision-Repair

• This mechanism is used to replace regions of damaged DNA up to 30 bases in length.

• Common examples of DNA damage include ultraviolet (UV) light, which induces the formation of cyclobutane pyrimidine-pyrimidine dimers, and

• smoking, which causes formation of• benzo[a]pyrene-guanine adducts.

• Ionizing radiation,cancer chemotherapeutic agents, and a variety of chemicals found in the environment cause base modification,

• strand breaks, cross-linkage between bases on opposite strands or between DNA and protein, and numerousother defects.

This gap is then filled in by a polymerase(δ/ε in humans) and religated.

Genetics of NER in Humans1- Xeroderma Pigmentosum Occurrence: 1-4/106 population Sensitivity: sunlight (ultraviolet)

Disorder: multiple skin disorders; malignancies of the skin neurological and ocular abnormalities Biochemical defect: early step of NERGenetic: seven genes (A-G), autosomal recessive

2- Cockayne’s SyndromeOccurrence: 1 per/ 106 population Sensitivity: sunlight

Disorder: arrested development ,

mental retardation,dwarfism, deafness, optic atrophy, intracranial calcifications skin cancer is increased 1000- to 2000-fold

Biochemical defect : NERGenetic: five genes (A, B and XPB, D & G)autosomal recessiveTranscription factor TFIIH

Genetics of NER in Humans

Summary

The tumor suppressor p53

• , a protein of MW 53 kDa, plays a key role in both G1 and G2 checkpoint control.

• Normally a very unstable protein, p53 is a DNA binding transcription factor, one of a family of related proteins, that is somehow stabilized in response to DNA damage,

• perhaps by direct p53-DNA interactions

• Increased levels of p53 activate transcription of an ensemble of genes that collectively serve to delay transit through the cycle.

• One of these induced proteins, p21CIP, is a potent CDK-cyclin inhibitor (CKI) that is capable of efficiently

• inhibiting the action of all CDKs.

• Clearly, inhibition of CDKs will halt progression through the cell cycle

• If DNA damage is too extensive to repair,

the affected cells undergo apoptosis (programmed cell death) in a p53-dependent fashion.