Pre Med III Genetics Guri Tzivion, PhD Extension 506 Summer 2015 Windsor University School of Medicine

Questions on Translation and Proteins?

From messenger RNA to protein: Translation The mRNA is translated by ribosomes as series of 3 letter codes designated codons. How many combinations are there?

The Genetic Code

Transfer RNA Transfer RNA molecules are short RNAs that fold into a characteristic cloverleaf pattern. Each tRNA has 3 bases that make up the anticodon. These bases pair with the 3 bases of the codon on mRNA during translation. Each tRNA has its corresponding amino acid attached to the 3’ end. Aminoacyl tRNA synthetases “charge” the tRNA with the proper amino acid.

Schematic model showing the binding sites on the ribosome P site (Peptidyl-tRNA binding site) E site (Exit site) mRNA binding site A site (Aminoacyl- tRNA binding site) Large subunit Small subunit EPA The assembled ribosome has one exit site and two tRNA-binding sites, designated A and P for aminoacyl and peptidyl-tRNA binding sites respectively. Only fMet-tRNA fMet can be used for initiation by the 30S subunits; all other aminoacyl-tRNAs are used for elongation by the 70S subunits.

Met GTP Initiator tRNA mRNA 5 3 mRNA binding site Small ribosomal subunit Start codon P site 5 3 Translation initiation complex E A Large ribosomal subunit GDP Met The initiation process involves the association of the mRNA, the initiator methionine-tRNA and the small ribosomal subunit. Several additional “initiation factors” -are also involved. The large ribosomal subunit then joins the complex. Initiation in prokaryotes

Polypeptide tRNA with amino acid attached Ribosome tRNA Anticodon 3 5 mRNA Amino acids Codons Elongation - Amino acids are added one by one to the preceding amino acid - Elongation factors facilitate - codon recognition - peptide bond formation - translocation

3 The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. The two ribosomal subunits and the other components of the assembly dissociate. Release factor Stop codon (UAG, UAA, or UGA) Free polypeptide When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. Termination

Pre Med III Genetics Class 9 DNA: Structure, Replication and Regulation of Gene Expression 4. Gene mutations and DNA repair

DNA Damage and Mutations

Mutations, definition Mutation - any change made to the DNA sequence or chromosome structure. 1) Inherently can either have beneficial or negative effect or have no significance. For example, they can lead to disease or death or promote evolution by generating new alleles. 2) They are permanent – can’t be removed or repaired (damage versus mutation) 3) They do not occur selectively and are random

Mutations are classified using several categories: 1) Size: Mutations can either involve large portions of chromosomes or small regions: a) Chromosomal mutations: Large segments of chromosomes are deleted, inverted, moved, or duplicated. b) Gene mutations: Smaller changes in the DNA sequence - one or few nucleotides 2) Cause: a) Natural biochemical events (spontaneous mutations) b) Induced mutations: Chemical, radiation, viral, etc.

Spontaneous mutations: mutations that occur in the absence of known mutagens  Uncorrected errors that occur during DNA replication, repair or recombination Spontaneous lesions that occur in the DNA molecule under normal physiological conditions and that are not repaired by the cell’s DNA excision repair processes

3) The type of the cell that contains the mutated DNA: a) Somatic mutations, arise in the DNA of somatic cells (normal diploid cells), do not pass to the next generation. b) Germ-line mutations: arise in the DNA of gamete- forming tissue (those cells that produce sperm and eggs). Are transmitted to the offspring and pass to the future generations.

Other Mutation Categories: 1. Lethal mutations: cause embryonic lethality 2. Conditional mutations: only express under certain environmental conditions, for example heat-sensitive mutations. 3. Suppressor mutations: indirectly reverse the effect of previous mutation (occur at different sites than the original mutations).

Small gene mutations come in 3 main varieties: A. Base-pair substitutions: One nucleotide is changed to a different nucleotide. Three possible outcomes on the amino acid sequence: 1.Silent mutation: No effect, usually when the change occurs in the 3 rd nucleotide of a codon. 2.Missense mutation: The change causes the wrong amino acid to be inserted. Can be Natural mutation if the new amino acid has a similar structure to the previous aa. 3.Nonsense mutation: Change turns the codon into a stop codon. Results in a truncated protein.

A. Base-pair substitutions: 1.Silent mutation: No effect, usually when the change occurs in the 3 rd nucleotide of a codon. 2.Missense mutation: The change causes the wrong amino acid to be inserted. Can be Natural mutation if the new amino acid has a similar structure to the previous aa. 3.Nonsense mutation: Change turns the codon into a stop codon. Results in a truncated protein.

B. Insertion/deletion: An extra nucleotide gets added or removed, causing a frame-shift. All amino acids after the insertion/deletion site will be altered!!

C. Expansion of trinucleotide repeats (TNRE) Some loci contain a series of trinucleotide repeats next to a gene or inside the gene (e.g: CAGCAGCAG...) The mutation causes an increase in the copy number. Not clear what causes the increase: maybe abnormal DNA structure causes the DNA pol to slip and copy the section twice? Such expansion often leads to disease: If in a gene, expansion increases the # of amino acids If next to a gene, can trigger methylation of gene TNRE disorders usually get worse each generation Expansion grows  worse symptoms

Types of DNA Damage 1.Deamination: (C  U and A  hypoxanthine) 2.Depurination: purine base (A or G) lost 3.T-T and T-C dimers: bases become cross-linked, T-T more prominent, caused by UV light (UV-C (<280 nm) and UV-B ( nm) 4.Alkylation: an alkyl group (e.g., CH 3 ) gets added to bases; chemical induced; some harmless, some cause mutations by mispairing during replication or stop the polymerase altogether

5. Oxidative damage: guanine oxidizes to 8-oxo- guanine, also causes single and double strand DNA breaks. 6. Replication errors: wrong nucleotide (or modified nt) inserted 7. Double-strand breaks (DSB): induced by ionizing radiation, transposons, topoisomerases, homing endonucleases, mechanical stress on chromosomes, or a single-strand nick in a single-stranded region (e.g., during replication and transcription)

DNA is damaged by Alkylation, Oxidation and Radiation Often mispaired with thymine G:C –A:T Reactive oxygen species O 2-, H 2 O 2, OH

Multiple DNA repair pathways Base excision repair (BER) Nucleotide excision repair (NER) Mismatch repair (MR) DNA strand cross link repair Homologous recombination (HR) Non-homologous end joining (NHEJ) Level of damage

Excision repair Two major types of excision repair: I.Base-Excision repair: Remove abnormal or modified bases from DNA. II. Nucleotide-Excision Repair: Remove larger defects like thymine dimers. Base- Excision Repair: Initiated by a group of enzymes called DNA glycosylases (recognize abnormal bases in DNA). The glycosylases cleave glycosidic bonds between the abnormal base and the 2-deoxyribose.

Base Excision Repair There are different DNA glycosylases, for different types of damaged bases. AP endonuclease recognizes sites with a missing base; cleaves sugar-phosphate backbone. Deoxyribose phosphodiesterase removes the sugar-phosphate lacking the base.

Nucleotide Excision Repair: Removes bulky DNA lesions that distort the double helix. An enzyme complex recognizes the distortion resulting from the damage. Additional enzymes separate the two nucleotide strands at the damaged region and single strand binding proteins stabilize the separated strands. The sugar phosphate backbone is cleaved on both sides of the damage. Part of the damaged is peeled away and the gap is filled by DNA polymerase and sealed by DNA ligase.

Nucleotide Excision Repair

Base excision repairNucleotide excision repair

Mismatch repair Many incorrectly inserted nucleotides detected by proofreading are corrected by mismatch repair. Enzymes cut out the distorted section of the newly synthesized strand of DNA and replace it with new nucleotides. The proteins that carry out this in E.coli differentiate between old and new strands of DNA by the presence of methyl groups. Adenine nucleotides in GATC sequence is methylated.

The mismatch repair complex brings the mismatched bases close to the methylated GATC sequence and the new strand is identified. Exonucleases remove nucleotides on the new strand between the GATC sequence and the mismatch. DNA polymerase replaces the nucleotides, correcting the mismatch and DNA ligase seals the nick in the sugar phosphate backbone.

Mismatch repair system

The nucleotide excision repair system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template and repair the errors Transcription-coupled DNA repair

DSBs are created biologically by the protein SPO-11 as the highly regulated initiation of meiotic recombination. Double stranded breaks Ionizing radiation Replication errors Oxidating agents environment endogenous Only used shortly after DNA replication, during interphase

Double Strand DNA Break Repair The repair of double strand breaks is an important mechanism for repairing damaged DNA induced by ionizing radiation or oxidative free radicals. It is also for example, part of the physiological process of immunoglobulin gene rearrangement. Some chemotherapeutic agents can also cause double stranded DNA breaks or prevent their repair.

Two proteins are involved in the non-homologous rejoining of DS breaks: Ku, that binds to free DNA ends and has a helicase activity, and DNA-dependent protein kinase (DNA–PK). DNA–PK also has binding sites for DNA free ends and allows the approximation of the 2 separated ends. Ku unwinds the DNA and the unwound approximated DNA forms base pairs. The extra nucleotide tails are removed by an exonuclease and the gaps are filled and closed by DNA ligase.

Genetic diseases associated with defects in DNA repair