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

DNA damage, repair & Mutagenesis 1 Mutagenesis Mutation Replication fidelity Mutagens: chemical & physical Mutagenesis: direct & indirect

Mutation Replication Fidelity Mutagens Mutagenesis

1-1 Mutation 1 Mutaagenesis Permanent, heritable alterations in the base sequence of DNA Reasons 1.Spontaneous errors in DNA replication or meiotic recombination 2.A consequence of the damaging effects of physical or chemical mutagens on DNA

TransitionTransition : Purine or pyrimidine is replaced by the other A  GT  C Transversion: a purine is replaced by a pyrimidine or vice verseTransversion A  T or C T  A or G G  T or C C  A or G Point mutation (a single base change) 1 Mutaagenesis

Noncoding DNA Nonregulatory DNA 3 rd position of a codon Silent mutation Coding DNA  altered Missense mutation Phenotypic effects No Coding DNA  stop codon  truncated protein Nonsense mutation Effects of a point mutation 1 Mutaagenesis Yes or No Yes

Insertions or deletions Frameshift mutations The translation of a protein encoded gene is frameshifted, then changed the C-terminal side of the mutation is completely changed. The addition or loss of one or more bases in a DNA region 1 Mutaagenesis

Examples of deletion mutations

1-2 Replication fidelity 1 Mutaagenesis Mutation relevant 1.Spontaneous errors in DNA replication is very rare, one error per base in E. coli.

Molecular mechanisms for the replication fidelity 1.DNA polymerase: Watson-Crick base pairing 2.3’  5’ proofreading exonuclease. 3.RNA priming: proofreading the 5’ end of the lagging strand 4.Mismatch repair 1.DNA polymerase: Watson-Crick base pairing 2.3’  5’ proofreading exonuclease. 3.RNA priming: proofreading the 5’ end of the lagging strand 4.Mismatch repair 1 Mutaagenesis

by E. coli polymerase Proofreading 1 Mutaagenesis

Mutagens 1 Mutaagenesis Mutation relevant Cause DNA damage that can be converted to mutations.

Physical mutagens High-energy ionizing radiation: X-rays and g-rays  strand breaks and base/sugar destruction Nonionizing radiation : UV light  pyrimidine dimers Chemical mutagens Base analogs: direct mutagenesis Nitrous acid: deaminates C to produce U Alkylating agents Intercalating agents Lesions-indirect mutagenesis 1 Mutaagenesis

Base analogs: derivatives of the normal bases incorporated in DNA, altering base pairing properties. Nitrous acid: deaminates C to produce U, resulting in G·C  A·U

Mutagenesis 1 Mutaagenesis The molecular process in which the mutation is generated. Note: the great majority of lesions introduced by chemical and physical mutagens are repaired by one or more of the error-free DNA repair mechanisms before the lesions is encounter by a replication fork

Direct mutagenesis The stable, unrepaired base with altered base pairing properties in the DNA is fixed to a mutation during DNA replication. 1 Mutaagenesis

5-BrU : G : A enol form Br OH H O Br Keto form H O AGCTTCCTA TCGAAGGAT AGCTBCCTA TCGAAGGAT 1.Base analog incorporation AGCTBCCTA TCGAGGGAT AGCTTCCTA TCGAAGGAT 2.1st round of replication AGCTBCCTA TCGAAGGAT AGCTCCCTA TCGAGGGAT 3.2nd round of replication A·T  G·C transition 1 Mutaagenesis

Indirect mutagenesis The mutation is introduced as a result of an error-prone repair. Translesion DNA synthesis to maintain the DNA integrity but not the sequence accuracy: when damage occurs immediately ahead of an advancing fork, which is unsuitable for recombination repair the daughter strand is synthesized regardless of the the base identity of the damaged sites of the parental DNA. 1 Mutaagenesis

E. coli translession replication: SOS response: Higher levels of DNA damage effectively inhibit DNA replication and trigger a stress response in the cell, involving a regulated increase (induction) in the levels of a number of proteins. This is called the SOS response. 1.Some of the induced proteins, such as the UvrA and UvrB proteins, have roles in normal DNA repair pathways. 2.A number of the induced proteins, however, are part of a specialized replication system that can REPLICATE PAST the DNA lesions that block DNA polymerase III. back 1 Mutaagenesis

Proper base pairing is often impossible and not strictly required at the site of a lesion because of the SOS response proteins, this translesion replication is error-prone. The resulting increase in mutagenesis does not contradict the general principle that replication accuracy is important (the resulting mutations actually kill many cells). This is the biological price that is paid, however, to overcome the general barrier to replication and permit at least a few mutant cells to survive.

DNA damage and repair Mutagen Completely repaired DNA damage (lesions) chemical reactivity of the bases Error-free Repairing mutations Indirect mutagenesis DNA damage, repair and mutagenesis minor or moderate Extensive, right before R eplication F ork (not repairable) Direct mutagenesis