10 TH WEEK DNA damage repair Mutagenesis Gihan

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

• DNA damage, repair & mutagenesis Mutation: replication fidelity, mutagens, mutagenesis DNA damage DNA lesions: oxidative damage, alkylation, bulky adducts DNA repair 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 Mutagenesis Mutagens

• 1 Mutaagenesis 1 -1 Mutation 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

• 1 Mutaagenesis Point mutation (a single base change) Transition : Purine or pyrimidine is replaced by the other A G T C Transversion: a purine is replaced by a pyrimidine or vice verse A T or C T A or G G T or C C A or G

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

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

Examples of deletion mutations

• 1 Mutaagenesis 1 -2 Replication fidelity Important for preserve the genetic information from one generation to the next Mutation relevant 1. Spontaneous errors in DNA replication is very rare, one error per 1010 base in E. coli.

1 Mutaagenesis 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 Mutaagenesis Proofreading by E. coli polymerase

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

1 Mutaagenesis 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

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

1 Mutaagenesis Mutagenesis 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

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

1 Mutaagenesis OH AGCTTCCTA TCGAAGGAT Br H : G O AGCTBCCTA TCGAAGGAT enol form 2. 1 st round of replication AGCTTCCTA TCGAAGGAT Br H : A O Keto form 5 -Br. U 1. Base analog incorporation AGCTBCCTA TCGAGGGAT 3. 2 nd round of replication AGCTBCCTA TCGAAGGAT A·T G·C transition AGCTCCCTA TCGAGGGAT

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 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 Uvr. A and Uvr. B 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 back DNA polymerase III.

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