Gihan E-H Gawish, MSc, PhD Ass. Professor Molecular Genetics and Clinical Biochemistry KSU 10 TH WEEK DNA damage, repair & Mutagenesis

Mutagenesis √ DNA damage, repair & mutagenesis DNA damage Mutation: replication fidelity, mutagens, mutagenesis DNA repair DNA lesions: oxidative damage, alkylation, bulky adducts Photoreaction, alkyltransferase, excision repair, mismatch repair, hereditary repair defects

2 DNA damage DNA damage DNA lesions: oxidative damage Alkylation bulky adducts

DNA lesions Oxidative damage Alkylation Bulky adducts 1.Occurs under Normal conditionOccurs under Normal condition 2.Increased byIncreased by ionizing radiation (physical mutagens) Alkylating agents (Chemical mutagens) UV light (physical mutagens) Carcinogen (Chemical mutagens) 2 DNA damage,

2 DNA damage 2-1DNA lessions An alteration to the normal chemical or physical structure of the DNA

The biological effect of the unrepaired DNA lesions Lethal (cell death) Physical distortion of the local DNA structure Blocks replication And/or transcription Mutagenic Allowed to Remained in the DNA A mutation could become fixed by direct or indirect mutagenesis Living cell Altered chemistry of the bases DNA lessions

Spontaneous DNA lesions 1.Inherent chemical reactivity of the DNA 2.The presence of normal, reactive chemical species within the cell 1.Deamination : C  UC  U methylcytosine  T, hard to be detected 2.Depurination : break of the glycosylic bond, non-coding lesion. 3.Depyrimidine back

Chemical reactivity of bases is responsible for some DNA lesion DNA lessions

deamination --ATGCTACG-- --TACGATGC-- --ATGUTACG-- --TACGATGC-- --ATG TACG-- --TACGATGC-- U --ATGCTACG-- --TACGATGC-- Uracil DNA glycosylase Cytosine deamination and repair back

2 DNA damage 2-2Oxidative damage DNA lesions caused by reactive oxygen species such as superoxide and hydroxyl radicals

Oxidation products 1. occurs under NORMAL conditions in all aerobic cells due to the presence of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and the hydroxyl radicals (OH). 2.The level of this damage can be INCREEASED by hydroxyl radicals from the radiolysis of H 2 O caused by ionizing radiation 2 DNA damage

2 DNA damage, 2-3Alkylation Nucleotide modification caused by electrophilic alkylating agents such as methylmethane sulfonate and ethylnitrosourea

Alkylated bases 1.Electrophilic chemicals adds alkyl groups to various positions on nucleic acids 2.Distinct from those methylated by normal methylating enzymes. alkylating agents

2 DNA damage 2-4Bulky adducts DNA lesions that distort the double helix and cause localized denaturation, for example pyrimidine dimers and arylating agents adducts These lesions disrupt the normal function of the DNA

Cyclobutane pyrimidine dimer Guanine adduct of benzo[a]pyrene Aromatic arylating agents Covalent adducts back DNA damage

3 DNA repair DNA repair 1-Photoreactivation 2-Alkyltransferase 3-Exision repair 4-Mismatch repair Hereditary repair defects

3 DNA repair 3-1: Photoreactivation Monomerization of cyclobutane pyrimidine dimers by DNA photolyases in the presence of visible light Direct reversal of a lesion and is error-free

3 DNA repair 3-2: Alkyltransferase Direct reversal of a lesion and is error-free Removes the alkyl group from mutagenic O 6 - alkylguanine which can base-pair with T. The alkyl group is transferred to the protein itself and inactivate it.

The response is adaptive because it is induced in E. coli by low levels of alkylating agents and gives increased protection against the lethal and mutagenic effects of the high doses DNA repair

3-3Nucleotide excision repair DNA repair 1.An endonuclease cleaves DNA a precise number of bases on both sides of the lesions (UvrABC endonulcease removes pyrimidine dimers) 2.Excised lesion-DNA fragment is removed 3.The gap is filled by DNA polymerase I and sealed by ligase

Base excision repair DNA glycolases cleaves apurinic or pyrimidine site DNA polymerase DNA ligase DNA repair cleaves N-glycosylic bond AP endonuclease 3’  5’ cleavage and & 5’  3’ synthesis

3 DNA repair 3-4: Mismatch repair A specialized form of excision repair which deals with any base mispairs produced during replication and which have escaped proofreading error-free

The parental strand is methylated at N 6 position of all As in GATC sites, but methylation of the daughter strand lag a few minutes after replication MutH/MutS recognize the mismatched base pair and the nearby GATC DNA helicase II, SSB, exonuclease I remove the DNA fragment including the mismatch DNA polymerase III & DNA ligase fill in the gap Expensive to keep the accuracy