Lecture 3.Mutations and DNA Reparation Myandina Galina Ivanovna professor, Ph.D.,Dr.Sc.

The mutant variants of one gene are allelic

The substitution - one nucleotide is replaced by another

A changes in DNA sequence are reflected in the changes of corresponding RNA or protein

DNA RNA Protein UUA Leu (wild type allele) UUA CUA Leu (silent ) UUA UAA stop (nonsense) UUA UUU Phe (missense) UUA AUA Ile (neutral)

Types of nucleotides’ alterations in DNA Substitutions are of two types: Transition – one purine is replaced by another purine, or one pyrimidine is replaced by another pyrimidine: GC → AT Transversion - one purine is replaced by a pyrimidine: A → C, G → T Frame shift mutation - deletion or insertion of nucleotides cause the break of the message downstream, it results in the production of an incorrect protein

Sickle-cell anemia is due to a substitution of CTT to CAT in gene of β-chain of hemoglobin

Formation of pyrimidine dimers by UV radiation

The radiation and chemicals can damage the DNA

Deamination of DNA bases by nitrous acids Adenine converts in hypoxanthine (HX), HX bonds with G, pair A -T is replaced by G - C. Cytosine becomes uracil, C-G is replaced A –U

Alkylation – adding the methyl-groups

Depurination – the loss of base in DNA strand It produces gaps in DNA strand The gap may produce a deletion or may be filled with incorrect base

Tautomerization causes the substitutions The uncommon configurations of nitrogen bases are called tautomers* When nitrogen base is in tautomeric form it cannot be linked to it’s complementary base: *Adenin forms a bond with C; Guanin* – with T

Bases’ analogues can be incorporated into DNA chain 5’-Bromuracil (BU) analogues of thymine, links usually with adenin 5’-BU has tautomeric form which links to guanine: Adenin = 5’-BU (usual); Guanin ≡ 5’-BU* (unusual)

Photoreactivation (light repair system) Photolyase (PR-enzyme) cleaves the bonds of dimers using the energy of visible light

Nucleotide excision repair (NER)

Excision repair (dark repair) endonucleases UvrA, B, C – make a nick in the affected strand DNA-polymerase I synthesizes new DNA strand Ligase seals the gap between the new strand and main DNA strand

Mismatch repair system Protein MutS - recognizes the mistake (G-T) Protein MutH cuts the incorrect strand Exonuclease I removes the DNA fragment DNA-polymerase fills the gaps Ligase seals the gap

Categories of DNA Repair Systems Photoreactivation is the process whereby genetic damage caused by ultraviolet radiation is reversed by subsequent illumination with visible or near-ultraviolet light. Nucleotide excision repair stages. The first stage involves recognition of the damaged region. In the second stage, two enzymatic reactions serve to remove, or excise, the damaged sequence. The third stage involves synthesis by DNA polymerase of the excised nucleotides using the second intact strand of DNA as a template. Lastly, DNA ligase joins the newly synthesized segment to the existing ends of the originally damaged DNA strand Base excision repair allows for the identification and removal of wrong bases, typically attributable to deamination—the removal of an amino group (NH2)—of normal bases as well as from chemical modification

Other categories of DNA Repair Systems Recombination repair, or post-replication repair, fixes DNA damage by a strand exchange from the other daughter chromosome. Because it involves homologous recombination, it is largely error free. Mismatch repair is a multi-enzyme system that recognizes inappropriately matched bases in DNA and replaces one of the two bases with one that "matches" the other. The major problem here is recognizing which of the mismatched bases is incorrect and therefore should be removed and replaced. SOS repair or inducible error-prone repair is a repair process that occurs in bacteria and is induced in the presence of potentially lethal stresses, such as UV irradiation. In this type of repair process, replication of the DNA template is extremely inaccurate.

xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin cancer incidence and premature agingxeroderma pigmentosum Cockayne syndrome: hypersensitivity to UV and chemical agentsCockayne syndrome Werner's syndrome: premature aging and retarded growthWerner's syndrome Bloom's syndrome: sunlight hypersensitivity, high incidence of malignancies (especially leukemias)Bloom's syndromemalignanciesleukemias Other diseases associated with reduced DNA repair function include Fanconi's anemia, hereditary breast cancer and hereditary colon cancerFanconi's anemiabreast cancercolon cancer Hereditary DNA repair disorders