Telomerases as drug targets. PHAR2811 Dale s lecture 6. Telomerases as drug targets. Oligodeoxynucleotide treatment

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1 Telomerases as drug targets PAR2811 Dale s lecture 6 Repair and Mutation Telomerase is active in between 80 and 90% of all cancers Targeting the RA component with antisense oligodeoxynucleotides and Rase (see following slides) Reverse transcriptase inhibitors; AZT, dideoxyguanidine Telomerases as drug targets Telomerase is active in between 80 and 90% of all cancers Inhibitors of the catalytic protein subunit G-quadruplex stabilisers..the th 3 overhang is G rich and tetra-stranded DA can form. This only happens with G-rich sequences. If this aberrant structure can be stabilised the end is blocked and telomerase activity is inhibited. 5 ligodeoxynucleotide treatment TTAGGG AAUCCCAAU Telomerase, containing an RA component. This enzyme was discovered by an Australian scientist, Elizabeth Blackburn. The sequence shown here is the tetrahymena sequence originally worked on by Elizabeth Blackburn. The human sequence is 11 nucleotides, rather than TTAGGG 5 3 TTAGGG 3 5 AAUCCCAAU 3 5 It is tricky to introduce these to the cell! 3 5 AAUCCCAAU 5 3 If oligonucleotides are introduced to the cell which are complementary to the RA sequence that binds to the telomere they will bind to the telomerase preventing it binding to the telomere. If oligonucleotides are introduced to the cell which are complementary to the RA sequence that binds to the telomere they will bind to the telomerase preventing it binding to the telomere. 1

2 5 3 TTAGGG 3 5 If you combine it with Rase you will then destroy the telomerase by degrading the RA. Rase degrades RA in DA:RA hybrids. Activation of the cell s endogenous Rase is being considered. 3 5 Repair Repair of DA is vital to the survival of the species. DA has the capability to repair itself, unlike RA. The extra copy provides the template and elaborate repair mechanisms have evolved to correct corruptions. Many errors at the time of replication are corrected by the 3 5 exonuclease activity of DA pols I & III. Repair There are corruptions to the sequence which occur after replication. An example. There are 3.2 X 10 9 purine nucleotides in the human genome. Each day ~ glycosidic bonds are cleaved from these purines in a given cell under physiological conditions. The conclusion: your cells contain some nasty little compounds. There are 130 genes which encode proteins responsible for repair in the human genome. UV damage (or the battle of the bulge) DA, when exposed to UV irradiation, forms pyrimidine dimers. Carbons 5 and 6 of two consecutive pyrimidines form covalent bonds with each other (cyclobutyl ring). These cyclobutyl bonds are ~1.6 Å which is much shorter than the distance between the bases (~3.4 Å). This causes a bulge in the double helix which prevents transcription or replication. This dimer has to go!! Pyrimidine dimers UV exposure 2

3 UV damage (or the battle of the bulge) There are a couple of ways of repairing this dimer. ne is to cleave the bonds directly, with an enzyme known as photolyase. This enzyme uses the energy from visible light to cleave the cyclobutyl bond. Photolyase is present in many prokaryotes and eukaryotes (not humans however). Photolyase: uses energy absorbed by visible light to break the covalent bond UV damage (or the battle of the bulge) Another more general type of repair is excision repair. The pyrimidine dimer, and a number of ajoining nucleotides are cut out by a set of enzymes responsible for nucleotide excision repair (ER). ER is carried out in E. coli by UvrA, UvrB and UvrC. UV damage (or the battle of the bulge) These enzymes seek out the bulge and cut out the offending nucleotides. The 11 to 12 oligomer is then removed by UvrD. DA pol I then comes and fills in the missing section, using the other strand as a template. The gap is sealed by DA ligase. ER Using its 5 to 3 polymerase and its proof reading 3 exonuclease activity DA pol I nucleotides 3 Chunk excised Filled in with DA pol I 3

4 Gap sealed with ligase Uracils in DA Uracil, which comes about from the spontaneous deamination of cytosine [or for that matter hypoxanthine, another base which comes about from the deamination of adenine and xanthine, derived from the deamination of guanine], does not belong in DA. A set of enzymes (base excision repair, BER) cleaves out the base at the glycosidic bond leaving an apurinic or apyrimidinic site (AP). Uracils in DA The deoxyribose is then excised by an AP endonuclease along with several ajoining nucleotides. The whole section is then filled in by DA pol I and DA ligase. Base excised Wrong base: corruption by deamination e.g. cytosine to uracil, adenine to hypoxanthine Apurinic or apyrimidinic site (AP) Extra nucleotides then removed The deoxyribose is removed by an AP endonuclease DA pol I and ligase come in to mop up De-purination When purine bases are cleaved at the glycosidic bond (as in the example in the introduction) the AP endonuclease again comes into action to remove the deoxyribose. The DA pol I and ligase then mop up. 3 4

5 Mutations A mutant gene is one that has a different sequence to the normal or wild type gene. This change is inheritable. There are a variety of mutations ti which h may or may not cause a change in the phenotype. The vast majority of mutations are neutral, having no effect (positive or negative) on the organism. Mutations In multicellular organisms for a mutation to be inheritable it must be present in the germline cells (meiotic). Mutations in somatic cells (mitotic) LY will not be inherited (cancer). Changes to the RA code (errors in transcription) are not inherited. Static Mutations Static mutations are those where the change in the code becomes a stable incorporation into the genome of the germline cells as well as all somatic cells in the organism (except RBC) This change is transferred to the next generation so the genome of the offspring is the same as the parent. Static Mutations The classic mutations such as sickle cell anemia, CF, PKU are much studied examples of this phenomenon. The ultimate expression of these mutations as a phenotype depends on the genetic information from both parents and epigenetic factors. Dynamic Mutations Examples here are the trinucleotide repeats (TR). The mutation increases (increasing number of repeats) in severity with each generation It also varies between tissues of the same organism. Dynamic Mutations This leads to genetic anticipation. The following generation will be more affected or the onset of the disorder will be earlier than the previous generation. The increase in the copy number of the repeat can occur at replication, repair or recombination whenever DA is being copied. 5

6 Dynamic Mutations The theories as to the mechanism(s) causing this type of mutation are many and varied (which means they won t be covered in this course). They all involve the formation of an aberrant loop structure when the DA strands are separated. The copying process somehow stutters then. The more repeats the more likely the loop. Types of Mutations Transversion: purine replaced by a pyrimidine or vice versa. This has implications for the 3-D shape of the helix. Transition: pyrimidine for pyrimidine or purine for purine e.g. A G,C T Silent mutations: altered codon still codes for the same amino acid (code redundancy) Frameshift: shifts the reading frame by adding or deleting base(s). Leads to a non-functional protein. Types of Mutations eutral mutation: altered codon codes for a functional similar amino acid no effect on the functionality of protein. Point mutation: single base pair change, can be a substitution, deletion, or addition. Missense: altered codon for functionally different amino acid these can be lethal onsense: mutation produces a stop codon truncated protein also dangerous. Types of Mutations Splice mutation: produces a splice site or removes one (only in eukaryotes) Temperature sensitive: mutation causes a change the protein function which is temperature sensitive. Usually the protein functions normally at lower permissible temperatures (<30 o C) but is inactive at higher temperatures (>40 o C). Reversion: a mutation which reverts to wild type, referred to as revertants. Leaky mutation: A mutation which doesn t affect the organism under normal conditions. It will show up in stressed conditions. Mutagenesis. Definition of mutagen: a physical or chemical agent which causes mutation to occur at a higher frequency. atural or spontaneous mutations: These are the mutations which occur at a normal background rate all the time. These mutations in the genome can arise naturally in the course of a cell s life. atural or Spontaneous Mutagenesis. The alternative tautomer of the base. If the base flips to the alternative tautomer at the time of replication the wrong nucleotide will pair up and by two rounds of replication we have a base pair switch. An error in replication which is not picked up by the proof reading activity of DA pol III will result in a mutation. Deamination, of cytosines (discussed earlier) or adenine to hypoxanthine. These can be corrected by specific repair mechanisms. Depurination (see above) can also be corrected by specific repair mechanisms. 6

7 xidative deamination of cytosine Deamination of adenine leads to base pairing with cytosine deoxyribose deoxyribose Cytosine Uracil Adenine ypoxanthine Tautomers of Adenine Tautomers of Adenine donor acceptor Adenine at p 7. The ring has a pka of ~4 so at p 7 the majority of the molecules are in this form : acceptor donor A very small proportion of the adenines will assume this tautomeric form at p 7 The major tautomeric form of Adenine at p 7 2 C 3 : Thymine Tautomers of Adenine The effect of a tautomeric change at replication The very very minor tautomeric form of Adenine at p 7 : 2 : This tautomer bonds to Cytosine A T *A C A T A-T C-G A-T T-A If the adenine (A *A) flips to the alternative tautomer at the time of replication, just as the new incoming nucleotide is selected it will base pair to cytosine, rather than thymine as the donor and acceptor are the other way around. Within another generation you have a GC instead of an AT. 7

8 Induced Mutagenesis. Intercalators, planar ring structures which slide in between the base pairs causing a disruption to the normal base stacking eg ethidium Bromide, acridine orange, actinomycin D. Alkylating agents, which methylate or ethylate bases and result in altered base pairing during replication e.g methylmethane sulfonate (MMS), nitrosamine. Intercalators Distance of a base pair, fits in nicely and separates the base stacking Induced Mutagenesis. Alkylating agents Alkylating agents, which methylate or ethylate bases and result in altered base pairing during replication e.g methylmethane sulfonate (MMS), nitrosamine. Anti-cancer drugs, used to treat brain tumours e.g. Temozolomide or temodal alkylates guanine residues at positions 6 and 7 and interferes with DA replication. Guanine alkylating agents 2 C methylguanine Induced Mutagenesis. Base altering agents eg nitrous acid converts amino groups to keto groups. Base analogues e.g. 5-Bromouracil which replace thymine but base pair to G (tautomeric shift due to Br). Base analogues 5-Bromouracil which replaces thymine but base pairs to G (tautomeric shift due to Br). 3 C Br Thymine 5-Bromouracil 8

9 Testing Mutagenesis: the Ames test A quick screening test for potential mutagenic compounds. A strain of Salmonella which has a defect in the histidine biosynthetic pathway is plated out, as a lawn, on a medium containing minimal is (just enough to keep the cells alive but not enough to sustain proliferation) Testing Mutagenesis: the Ames test The compound of interest is applied to a disc in the centre of the plate and the plate is incubated overnight. Different plates with increasing amounts of the compound are put up. Sometimes liver extract is applied also to check for cellular conversions Testing Mutagenesis: the Ames test If the compound is mutagenic it will cause a number of cells to revert to wild type and grow on the medium; the other cells can t because of the is defect. The more colonies forming around the disc the more mutagenic the compound. A non-mutagenic compound will have a few colonies scattered over the whole plate (spontaneous reversions). Ames Test Mutagenic Response Ames Test A mutagenic compound will typically have a linear dose response. # colonies Concentration of potential mutagen 9

10 The negative control A mutagenic compound 9. The Ames test relies on a number of factors. Which of the following is T an assumption required for the Ames test? A. Salmonella spontaneously mutate at a very fast rate B. Wild type Salmonella can synthesise histidine de novo (from scratch) C. Mutagenic compounds will only induce mutations which cause is reversion D. Salmonella deficient in histidine synthesis (Sal his-/-) can be maintained on minimal histidine medium without proliferation E. A visible colony on an agar plate is the result of cell proliferation from a single cell 9. The Ames test relies on a number of factors. Which of the following is T an assumption required for the Ames test? A. Salmonella spontaneously mutate at a very fast rate B. Wild type Salmonella can synthesise histidine de novo (from scratch) C. Mutagenic compounds will only induce mutations which cause is reversion D. Salmonella deficient in histidine synthesis (Sal his-/-) can be maintained on minimal histidine medium without proliferation E. A visible colony on an agar plate is the result of cell proliferation from a single cell 10