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 nucleated cells each with 3x 10 9 base pairs of DNA. If about 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 10 6 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 Becquerel discovered radiation in 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

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 O 2 -, H 2 O 2, OH. 3- Ionizing radiation 4- Chemicals It causes: Base-mispairing (i.e., oxoG can pair with C or A) Base alteration/damage

b: 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 Ames Brith:1928 Ames test: 1970 Can we detect Mutagen: Ames Assay For carcinogens, based on their mutagencity Salmonula having a mutant that in active an enzyme of the His biosynthetic 90% found in eukaryotic

DNA Damage, Repair, and Consequences Damaging agentConsequences Repair Process In hibition of: Replication Transcription Chromosome segregation Mutation Chromosom e aberration

DNA Repair Pathways 4.Recombinational repair - multiple pathways - double strand breaks and inter strand cross-links 5.Tolerance mechanisms - lesion bypass - recombination 1. Direct reversals 2. 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 - lesion bypass - recombination 5.Tolerance mechanisms - lesion bypass - recombination

Damage Recognized: Thymine dimers 6-4 photoproduct Gene Products Required: Photolyase Related disease: Photolyase not yet found in placental mammals TT Visible light TT 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 endonuclease to 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 Humans 1- Xeroderma Pigmentosum Occurrence: 1-4/10 6 population Sensitivity: sunlight (ultraviolet) Disorder: multiple skin disorders; malignancies of the skin neurological and ocular abnormalities Biochemical defect: early step of NER Genetic: seven genes (A-G), autosomal recessive

2- Cockayne’s Syndrome Occurrence: 1 per/ 10 6 population Sensitivity: sunlight Disorder: arrested development, mental retardation, dwarfism, deafness, optic atrophy, intracranial calcifications skin cancer is increased to 2000-fold Biochemical defect : NER Genetic: five genes (A, B and XPB, D & G) autosomal recessive Transcription 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.