PHAR2811 Dale’s lecture 6

Repair and Mutation

Telomerases as drug targets

Telomerase is active in between 80 and 90% of all cancers

• Targeting the RNA component with antisense oligodeoxynucleotides and RNaseH (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 3’ overhang is

G rich and tetra-stranded DNA 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.

Oligodeoxynucleotide treatment

5’ 3’

3’ 5’

TTAGGGAAUCCCAAU

5’3’

Telomerase, containing an RNA 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 9.

5’ 3’

3’ 5’

TTAGGG

AAUCCCAAU

5’3’

If oligonucleotides are introduced to the cellwhich are complementary to the RNA sequence that binds to the telomere they will bind to the telomerase preventing it binding to the telomere.

TTAGGGTTA

TTAGGGTTA

TTAGGGTTA

TTAGGGTTA

It is tricky to introduce these to the cell!

5’ 3’

3’ 5’

TTAGGG

AAUCCCAAU

5’3’

If oligonucleotides are introduced to the cellwhich are complementary to the RNA sequence that binds to the telomere they will bind to the telomerase preventing it binding to the telomere.

TTAGGGTTA

TTAGGGTTA

TTAGGGTTA

TTAGGGTTA

5’ 3’

3’ 5’

TTAGGG

5’3’

If you combine it with RNaseH you will then destroy the telomerase by degrading the RNA . RNaseH degrades RNA in DNA:RNA hybrids. Activation of the cell’s endogenous RNaseH is being considered.

TTAGGGTTA

TTAGGGTTA

TTAGGGTTA

TTAGGGTTA

Repair• Repair of DNA is vital to the survival of the

species. • DNA has the capability to repair itself,

unlike RNA. 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 DNA pols I & III.

Repair• There are corruptions to the sequence which

occur after replication. • An example. There are 3.2 X 109 purine

nucleotides in the human genome. Each day ~10 000 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)

• DNA, 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

UV damage (or the battle of the bulge)

• There are a couple of ways of repairing this dimer.

• One 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 (NER). NER 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. DNA pol I then comes and fills in the missing section, using the other strand as a template.

• The gap is sealed by DNA ligase.

NER

Chunk excised Filled in with

DNA pol I

DNA pol IUsing its 5’ to 3’polymerase and its proof reading 3’ exonuclease activity

nucleotides

3’OH

Gap sealed with ligase

Uracils in DNA• 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 DNA.

• 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 DNA• The deoxyribose is then excised by an AP

endonuclease along with several ajoining nucleotides.

• The whole section is then filled in by DNA pol I and DNA ligase.

Wrong base: corruptionby deamination e.g. cytosineto uracil, adenine to hypoxanthine

Base excised

Apurinic or apyrimidinic site (AP)

The deoxyribose is removed by an AP endonuclease

Extra nucleotides then removed

3’OH

DNA 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 DNA pol I and ligase then mop up.

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 which

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) ONLY will not be inherited (cancer).

• Changes to the RNA 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 (TNR).• 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 DNA is being copied.

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 DNA 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. AG,CT

• 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• Neutral 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

• Nonsense: 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 (<30oC) but is inactive at higher temperatures (>40oC).

• 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.

• Natural 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.

Natural 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 DNA 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.

Oxidative deamination of cytosine

N

N

NH2

O

deoxyribose

N

N

O

O

deoxyribose

NH3

Cytosine Uracil

Deamination of adenine leads to base pairing with cytosine

N

NN

N

NH2

NH

NN

N

O

Adenine Hypoxanthine

Tautomers of Adenine

N

NN

N

N

N

NN

N

N:

H

H

H

H

acceptor

donor

donor

acceptor

Adenine at pH 7. The ring N has a pKa of ~4 so at pH 7 the majority of the molecules are in this form

A very small proportion of the adenines will assume this tautomeric form at pH 7

Tautomers of Adenine

N:

N

N

N NH2

HN

N

O

O

CH3

The major tautomeric form of Adenine at pH 7 Thymine

Tautomers of Adenine

The very very minor tautomeric form of Adenine at pH 7 This tautomer

bonds to Cytosine

N

N

N

N :N

H

H

N:

N

H2N

O

The effect of a tautomeric change at replication

A

T

*A

T

C

A

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.

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, 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 DNA replication.

Alkylating agents

NH

NN

N

O

NH2

CH3

NH

NN

N

O

NH2

alkylating agents

Guanine O6-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).

N

N

O

O

NH

N

O

O

BrH3C

Thymine 5-Bromouracil

H

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 His (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 His 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

• A mutagenic compound will typically have a linear dose response.

Concentration of potential mutagen

# colonies

Ames Test

The negative control A mutagenic compound

9. The Ames test relies on a number of factors. Which of the following is NOT 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 His 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 NOT 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 His 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