4.) DNA Repair Flashcards
What nucleotide base can be modified by one chemical reaction to form another nucelotide base?
Cytosine —> Uracil
Deamination reaction.
What is de-amination?
This is the removal of an amine group from an organic molecule, usually to be replaced by a carbonyl/hydroxy group.
What nucleotide is easily susceptible to epigenetic mutation?
5-methylcytosine, which is easily converted to thymine through deamination.
What are three common causes for spontaneous alteration that will require DNA repair?
- ) Spontaneous oxidative damage
- ) Hydrolytic attack (depurination or deamination)
- ) Uncontrolled methylation by S-andeosylmethionine
Induction of UV light onto DNA often results in the formation of…
Pyrimidine dimers (thymine usually), and subsequently a bend/kink.
What are the 4 repair mechanisms that we went over in this lecture?
- ) Mismatch repair
- ) Base excision repair
- ) Nucleotide-excision repair
- ) Homologous recombination
True or False: The mutations that we are discussing in this lecture are mutations in DNA replication.
False. These are mutations that occur after replication has taken place.
At what point would you find hemimethylated DNA?
Shortly after replication, the template strand is methylated whereas the daughter strand is not.
What enzyme is responsible for methylation of DNA?
Dam methylase
In methylation and mismatch repair what protein recognizes the GATC hemimethylated sequence? What protein detects the mismatched base pair?
MutH (req ATP)
MutS (req ATP)
Describe the process of methylation and mismatch repair.
There is a short period of time after replication when the daughter strand is unmethylated. The MutS protein runs along the DNA to find any mismatched base pairs. At the site of mutation, the MutS protein associates and will now form a complex with MutL. The DNA is cinched from both sides through the MutS an MutL complex until the complex runs into a methylation (detecting the template strand). MutH protein will bind to either side of the MutL and MutS complex. The structure looks like a loop at this point.
MutH will then cleave at the 5’ site of the G base on the unmethylated strand at the most proximal methylated site. This knick in the DNA is the point of excision repair. Depending on the side of the knick, you will either have a 5’—>3’ exonuclease or a 3’—>5’ exonuclease that will remove a portion of the daughter strand up to the mismatched based pair. DNA polyIII and the SSB (single stranded binding protien) will resynthesize the cleaved portion of the daughter strand and include the correct base pair.
What is the most stable and standard of the repair mechanisms?
Methylation and mismatch repair. (note jsut something he mentioned in class, probably won’t be on the test).
When would nucleotide excision repair be used?
When a portion of DNA is damaged by UV radiation (or carcinogens from cigarette smoking), forming a dimer.
What is depurination?
This is the complete removal of a nucleotide base from its sugar residue.
Nucleotide deletions often result from what type of chemical modification?
Depurination
Nucleotide substitution mutations often result from what type of chemical modification?
Deamination
When would base excision repair be used?
When a single nucleotide base is damaged.
*Often occurs with cystine since it easily deaminates to uracil.
Describe the process of base excision repair.
DNA glycosylase detects the damaged base and will cleave the nucleotide base from the sugar backbone. An AP endonuclease then cleaves the phosphodiester bond near the 5’ end. DNA polymerase I then starts synthesis at the free 3’ hydroxyl removing and replacing a portion of the damaged strand. Ligase then connects the knick that is formed after DNA poly I dissociates.
Describe the process of nucleotide excision repair.
DNA lesion forms due to the mutation. Excinuclease will cleave a phosphodiester bond upstream and downstream from the lesion on the damaged strand (29 units long in humans, 13 in bacteria). DNA helicase then removes the damaged portion of DNA. DNA polymerase (I in E. Coli, epsilon in humans) will resynthesize that portion of the strand. Ligase will then fill in the knick.
Non-homologous end-joining and homologous recombination are repair mechanisms that are used when…
There is an accidental double stranded break in DNA
What is the most common form of repair for double stranded breaks? What is the problem with this type of repair mechanism?
Non-homologous end-joining. It results in a deletion of DNA.
Describe the process of non-homologous end-joining.
Double stranded cleaved ends are recognized by Ku heterodimers. These recruit other proteins that will process the DNA to make blunt ends and then ligate them together.
What is the only double stranded break repair mechanism that we use in somatic cells that aren’t dividing?
Non-homologous end-joining.
When is the only time that homologous recombination repair of a double stranded break will occur?
S phase or early G2 phase. Need sister chromatids to act as templates
