Repair of DNA Flashcards
Discuss the fidelity of replication
There are many proteins that are involved in DNA replication- this is to maintain near perfect fidelity. A high replication accuracy arises from 5 sources:
> Cells maintain balanced levels of dNTPs - if a dNTP is present at high levels it is more likely to be misincorporated and if a dNTP is present at low levels its more likely to be replaced by the one present at high levels
> The polymerase reaction has fidelity- it occurs in two stages: a binding step in which the incoming dNTP pairs with the template while the enzyme is in open conformation, so no catalysis is occurring and then when catalysis takes place in a closed conformation.
> The 3’-5’ exonuclease activity in pol I and III detects and eliminates any errors made by the polymerase activity.
> The 5’-3’ exonuclease activity of pol I removes RNA primers
> Many enzyme systems that function to repair errors in newly synthesized DNA and any damage that occurs after synthesis.
Provide a brief explanation of DNA damage
DNA damage poses a continuous threat to genomic integrity. Cells have evolved DNA repair enzymes and repair polymerases that are complex. If DNA damage is severe it can be irreversible and can lead to a loss of genetic information and cell death. When DNA is repaired it ,ay not be perfect and this can lead to a mutation. Mutations that occur in somatic cells usually have no effect, unless they contribute to malignant transformation(cancer). Mutation that occur in germline cells are passed on to future generations.
Differentiate between spontaneous and induced mutations
Spontaneous mutations occur as a result of natural processes in cells, e.g. DNA replication errors. Induced mutations occurs as the result of interaction of DNA with an outside agent that causes the DNA damage.
What are the two major classes of mutations
The simplest type of mutation is a nucleotide substitution and mutations that alter a single nucleotide are called point mutations.
What are the different types of nucleotide substitutions
Transitions- when a pyrimidine is substituted with another pyrimidine or when a purine is subed with another purine.
Transversions- when a pyrimidine is subed with a purine or vice versa.
They can both lead to silent, missense or nonsense mutations.
Define synonymous mutations or silent mutations, nonsynonymous or missense mutations and a nonsense mutation
Mutations that change nucleotide sequence without
changing amino acid sequence
Nucleotide substitutions in protein-coding regions that do result in changed amino acids:
May alter the biological properties of the protein.
– Sickle cell anemia is an AT→TA transversion
– Glutamic acid codon in the β-globin gene replaced by a valine codon
A nucleotide substitution that creates a new stop codon:
– Causes premature chain termination during protein synthesis.
– Nearly always a nonfunctional product
Define indels
If the length of an insertion or deletion is not an exact
multiple of three nucleotides, this results in a shift in the
reading frame of the resulting mRNA.
– Usually leads to production of a nonfunctional protein.
Define DNA damage
DNA damage is any change to the double helical structure. These can result in:
> single base changes: affect the DNA sequence and has a minor effect on the overall structure of DNA
> structural distortion: an example is the induction of pyrimidine dimers by UV irradiation or intercalating agents that can cause indels during DNA replication
> DNA backbone damage: these include abasic sites(the loss of the nitrogenous base) and double-strand DNA breaks(the most severe form of DNA damage)
What are the 3 categories of DNA repair mechanisms
Direct reversal of damage, the removal of damaged DNA and replacement with undamaged DNA and mechanisms that bypass the damaged DNA(translesion DNA synthesis)
Provide 2 examples that involves the direct reversal of damaged DNA
The reversal of T-T dimers by DNA photolyase and the reversal by alkyltransferases
Explain the direct reversal of TT dimers by DNA photolyase
UV radiation induces the formation of a cyclobutane ring between adjacent thymine residues on the same DNA strand to form an intrastrand thymine-thymine dimer. The TT dimer distorts the double helix and can block replication and transcription. UV radiation can also induce dimers between cytosine and thymine. In most organisms, UV radiation damage to DNA can be directly repaired by light-absorbing enzymes called photoreactivating enzymes or DNA photolyases. DNA photolyases uses energy from near UV to blue light to break the covalent bonds holding the two adjacent pyrimidines together. DNA photolyase has 2 cofactors: a pigment that absorbs UV or blue light and fully reduced flavin dinucleotide(FADH-). The pigment absorbs UV or blue light, transfers the excitation energy to FADH-, which transfers an electron to the pyrimidine dimer already bound to the enzyme. The TT dimer is flipped out of the helix and brought close to FADH-. An electron is transferred from FADH- and the TT dimer is split. Photolyases are an ancient and efficient way to repair UV damaged DNA. However placental animals(including humans) do not have a photo reactivation pathway.
Explain damage repair by alkyltransferases
The alkylation of guanine at the oxygen atom results in O6-Methylguanine. The formation of these derivatives is highly mutagenic because during replication they cause the incorporation of thymine instead of cytosine. It is repaired by O6-alkylguanine-DNA alkyltransferase- this directly transfers the alkyl group to its own Cys residue. The reaction inactivates the protein, so strictly speaking it is not an enzyme.
Describe the steps involved in excision repair and the 2 types of excision repair mechanisms
These repair mechanisms have similar steps: recognition of damaged DNA, the removal of damaged DNA through excision, The replacement of DNA by DNA polymerases and the gaps sealed by DNA ligase.
The nucleotide excision repair(NER) repairs bulky DNA lesions such as structural distortions and base excision repair (BER) repairs nonbulky regions involving a single base.
Describe nucleotide excision repair
NER is a DNA repair mechanism that is found in all cells that eliminates damage to dsDNA by excising an oligonucleotide containing the lesion and filling in the resulting single-strand gap. This system is activated by a helix distortion rather than by recognition of a particular group. In humans NER is a defense against 2 important carcinogens- sunlight and cigarette smoke. In E.coli NER is an ATP-dependent process carried out by UvrA, UvrB, UvrC proteins. This system is called UvrABC endonuclease- it cleaves the damaged DNA strand at the 7th and at the 3rd or 4th phosphodiester bonds from the lesions 5’ and 3’ sides. The excised oilgonucleotide is displaced by the binding of UvrD and replaced by the actions of Pol I and DNA ligase.
Explain what occurs if the NER mechanism is genetically defective
This means that the DNA repair mechanism is not functioning. In humans a rare inherited disease called Xeroderma pigmentosum is mainly characterized by the inability of skin cells to repair UV-induced DNA lesions. Individuals with this autosomal recessive condition are extremely sensitive to sunlight. During infancy they develop skin changes- dryness, excessive freckling etc. They often develop fatal skin cancers at at a 2000-fold greater rate than the normal population. If the DNA contains a lesion, but needs to transcribe to RNA, an urgent response is required to repair the DNA.RNA polymerase cannot transcribe through DNA damage so it will stop and give time to the DNA repair system to repair. The DNA system that is recruited is transcription-coupled repair(TCR)- which only repairs the strand that needs to be transcribed. Cockayne syndrome is an inherited disease caused by defective TCR. Individuals are hypersensitive to UV radiation, exhibit stunted growth, but have a normal incidence of skin cancer.
