Week 4: DNA repair Flashcards
What percentage of random changes every day accumulate permanent mutations?
0.02%
What is depurination?
Purine bases lost by cells (18,000 a day)
Because their N-glycosyl linkages to deoxyribose is hydrolysed
Name the random change that occurs to cytosine molecules at the rate of 100 bases per day
Deamination of cytosine to uracil
How are DNA bases occasionally damaged?
Encounters with reactive metabolites produced by the cell including reactive forms of oxygen and the high energy methyl donor S-adenosymethionine
What mutations can ultraviolet mutations cause?
Can produce a covalent linkage between two adjacent pyrimidine bases in DNA e.g to form thymine dimers
What would happen if uncorrected DNA is replicated?
- Deletion of one or more base pairs
Or - Base pair substitution in the daughter DNA chain
The mutations would then be propagated throughout subsequent cell generation
How is h DNA helix suited for repair?
It carries two separate copies of all genetic information (one in each of its two strands)
If one strand is damaged the complementary strand is generally used to restore the correct nucleotide sequence of the damaged strand
Why can organisms with large genomes not afford to encode their genetic information in any molecule other than the DNA double helix?
Because once damaged the chance of a permanent nucleotide change occurring in single stranded genomes is very high
What happens in two of the most common pathways of DNA repair?
- The damage is excised
- Original DNA sequence is restored byDNA polymerase that uses the undamaged strand as its template
- A remaining break in the double helix is sealed by DNA ligase
What is the function of DNA glycolases?
Recognition of a specific type of altered DNA and catalyse its hydrolytic removal from its sugar
Travel along the DNA using base flipping to evaluate the status of each base
What is base excision repair?
Involves DNA glycosylases that recognise a specific type of altered DNA and catalyse its hydrolytic removal. This includes removal of :
- deaminated C’s or A’s
- different types of alkylated or oxidised bases
- bases with opened rings
- bases in which carbon- carbon double bond has been converted to a single bond
How is an altered base detected within the context of the double helix?
An enzyme mediated ‘flipping out’ of the altered nucleotide from the helix which allows the DNA glycosylase to probe all faces of the base for damage
What is the ‘missing tooth’ created by DNA glycosylase action recognised by?
AP (apurunic/ apryrimidinic) endonuclease
Signifies that the enzyme cleaves within the polynucleotide chain which cuts the phosphodiester backbone after which the resulting gap is repaired
Depurinations leave a deoxyribose regardless with a missing base. What are they directly repaired beginning with?
AP endonuclease
Explain nucleotide excision repair
This mechanism can repair the damage any large change in the structure the the DNA double helix.
- A large multienzyme complex scans the DNA for a distortion rather than for a specific base change
- Once it finds a lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion
- A DNA helicse peels away the single strand oligonucleotide containing the lesion
- The large gap produced is repaired by DNA polymerase and DNA ligase
Give examples of large lesions
- those created by covalent reaction of DNA bases with larger hydrocarbons
- various pyrimidine diners caused by UV radiation
What is the function of AP nuclease?
Recognises any site in the DNA helix that contains deoxyribose sugar with a missing base
What is an alternative to base and nucleotide excision repair?
Direct chemical reversal of DNA damage
What is direct chemical reversal of DNA damage usually employed?
For highly mutagenic or cytotoxic lesions
Give an example of direct chemical reversal of DNA damage
- The alkylation lesion O6- methylguanine has its methyl group removed by direct transfer to a cysteine residue in the repair protein itself, which is destroyed in the reaction
- Methyl groups in the alkylation lesions 1-methyladenine and 3-methyl cytosine are burned off by an iron dependent demethylase, with release of formaldehyde from the methylated DNA and regeneration of a native base
How do cells direct DNA repair to the sequences that are most urgently needed?
They link RNA polymerase to the nucleotide excision pathway.
RNA polymerase stalls at DNA lesions and through the use of coupling proteins directs the excision repair machinery to these sites
What happens to the stalled RNA polymerase when it reaches a lesion in bacteria?
(where the genes are relatively short)
RNA polymerase can be dissociated from the DNA, the DNA is repaired, the gene is transcribed from the beginning
What happens to the stalled RNA polymerase when it reaches a lesion in eukaryotes?
(Where the genes are relatively long)
A more complex reaction is used to back up the RNA polymerase, repair the damage and then restart the polymerase
What caused by a defect in transcription coupled excision repair?
Cockayne syndrome
What do individuals who have Cockayne syndrome suffer from?
- growth retardation
- skeletal abnormalities
- progressive neural retardation
- sever sensitivity to sunlight
How is hypoxathine produced?
Deamination of A
What is the simplest purine base capable of pairing with C?
Hypoxathine
Why does DNA have T instead of U?
The repair system would not be able to distinguish between a deaminated C from a naturally occurring U
What is employed when DNA suffers heavy damage and the normal repair mechanisms are insufficient to cope with it?
Translesion polymerase
Human cells have 7*
Why are translesion polymerases not as accurate?
- They lack exonucleic proofreading activity
- Many are less selective than replicative polymerase in choosing which nucleotide to incorporate initially
Each translesion polymerase is give the chance to add only one or a few nucleotides before the highly accurate replication polymerase resumes DNA synthesis
What causes DNA change?
- spontaneous
- endogenous factors
- exogenous factors
What are the two main types of spontaneous DNA damage?
- hydrologic depurination
- hydrolytic deamination of bases
What is formed when adenine is deaminated?
Forms hypoxanthine
What is formed by the deamination of guanine?
Xanthine
What are some endogenous factors?
Endogenous factors are produced by the metabolism of the cell
- superoxide O2-
- Hydrogen peroxide H202
- Hydroxyl radicals OH
What are some exogenous factors?
- high energy radiation (UV,X-Ray and gamma radiation)
- organic compounds
What is the relationship between wavelength and energy?
Shorter wavelength= highest energy In order of longest wavelength UV-A UV-B UV-C
What part of the DNA absorbs UV radiation
Aromatic rings
X-ray wavelengths (0.01-10 nm), energy >
124eV
Gamma radiation (<0.01nm) energy >
1,000,000 eV
What does energy transfer lead to?
Ionisation and radical formation.
What are the host harmful species arising from water and oxygen
OH- and O2 radicals
What do alkylating agents do?
Transfer alkyl groups (methyl, ethyl etc) on electron rich atoms
What are electron rich atoms found in DNA?
Nitrogen
Oxygen
What is cis platin?
What does it do?
A cancer drug
It blocks transcription and DNA replication
Forms cross-links
What are the consequences of DNA modification?
- Modifications of bases can change base pairing
- changed base pairing can result in a mutation
- double strand breaks can lead to gene rearrangement and loss of numerous genes
- mutations are inherited
- crosslinking of DNA strands inhibits transcription, translation and can kill a cell
What are the main types of mutations?
- point mutation= change of a bp
- deletion= one or several bp are lost
- insertion= one or several by are inserted
What is a silent mutation?
No consequences for the protein sequence
What is a nonsense mutation?
Protein sequence changed
Stop codon prematurely added
What can mutations in promoter/activator regions do?
Can modify gene expression and regulation
What can mutations in exons/ introns Boundaries do?
Disrupt the reading frame of a protein
How do translesion polymerases pose risks to the cell?
- responsible for most of the base substitution and single nucleotide deletion mutations that accumulate in genomes
- they generally produce mutations when copying damaged DNA
- they create mutations create mutations, at a low level, on undamaged DNA
What would happen if major lesions (sites where both strands of the double helix are broken) were left unrepaired?
They would quickly lead to the breakdown of chromosomes into smaller fragments and loss of genes when the cell divides
What is non-homologous end joining?
A fairly inaccurate method of double stranded break repair that predominates in humans
Broken ends are simply brought together and rejoined by DNA ligation, generally with the loss of nucleotides at the site of joining
Usually takes place when cells have not yet duplicated their DNA
Where is nonhomologous end joining a common mechanism for repair?
In mammalian somatic cells
What danger is presented by nonhomologous cell joining as a mechanism for repair?
There seems to be no mechanism to ensure that the two ends being joined together were originally next to each other in the genome this can cause unfavourable rearrangements:
One broken chromosome becomes covalently attached to each other which can result in chromosomes with two centromeres and chromosomes lacking centromeres altogether
What is the function of telomeres?
Specialised structures that prevent the natural ends of chromosomes from being mistaken from broken DNA and “repaired”
What is homologous recombination?
A more accurate method of double stranded break repair used only during and shortly after DNA replication (in S and G2 phase)
The DNA is repaired using the sister chromosome as a template
Which method of double stranded DNA repair restores the original DNA
Homologous recombination
In non-homologous end joining the initial degradation of the broken DNA ends is important because the nucleotides at the site of the initial break are often damaged and cannot be ligated thus altering the original DNA sequence when repairing a broken chromosome
What protein plays a central role in homologous end joining?
Ku protein
A heterodimer that recognised then grasps the broken chromosome ends
What could cause a delay in the cell cycle?
The detection of DNA damage which can block entry from G1 to S phase, it can slow S phase once it has begun and it can block the transition from G to M phase. These delays facilitate repairs by providing the time for the repair to reach completion
What is the function of the ATM protein?
It is a large kinase needed to generate the intracellular signals that sound the alarm in response to many different types of spontaneous DNA damage
What is the disease caused by the absence of the ATM protein?
Ataxia telangiectasia (AT) Symptoms: - neurodegeneration - predisposition for cancer - genome instability
What is formed when cytosine is deaminated?
Uracil
