Lecture 5: Mutability, DNA Damage and Repair Flashcards
1Name some sources that lead to mutations in the genome.
- Inaccuracy in DNA replication (Spontaneous mutagenesis)
- Due to tautomerization
- Replication errors that were not corrected during.
proofreading process
- Chemical Damage to the DNA (Induced mutagenesis)
- Spontaneous damage such as the loss of some bases
- Natural or unnatural mutagens
What are some consequences of replication errors?
- The genome can incur mutations which can alter the coding sequence of the gene or its regulatory sequences. These mutations typically only manifest in the progeny of the cell.
- DNA lesions can occur which are chemical alterations in the DNA. This can prevent the DNA’s use as a template for replication and transcription. This type of lesion will have immediate effects on the cell function and the survival of the cell
What are some actions that can be taken by cellular machinery in order to help reduce and prevent replication errors?
- Detecting the errors early on during the process of synthesis and helping correct the damage done to the DNA (proofreading process)
- Repair of the lesions and if its possible also restore the original DNA sequence.
What are the three types of mutations that you can give an example of?
- Substitution mutation
- Insertion of nucleotide(s)
- Deletion of nucleotide(s)
Explain the two types of substitution errors.
- Transition substitution error:
- Pyrimidine to Pyrimindine OR Purine to Purine
- Eg. T to C OR A to G
- Transversion substitution error:
- Pyrimidine to Purine OR Purine to Pyrimidine
- T to G or A
- A to c or T
What are point mutations?
These are mutations that alter a single nucleotide, they are known as SNPs (Single nucleotide polymorphism)
What are Chromsomal Aberrations?
Structural changes in the structure of the chromosome.
For example, long range rearrangements such as insertions, deletions, translocations, duplications
Explain the term “Hotspots” in regards to Mutations in the DNA
Hot spots are specific genomic sites where the mutation rate is higher. An example of this is DNA microsatellites which are a mutation-prone sequence as It contains di-, try-, or tetra- nucleotide repetitive DNA sequences.
They typically are widely scattered in the chromosomes of humans or other eukaryotes. The reason it is more common in repetitive DNA sequences is because replication machinery has difficulty copying repeats accurately, and this can result in frequent “slippage”. This leads to an increase or a decrease in the number of copies of the repeated sequence.
These hotspot regions can often lead to TNRE diseases such as Huntington’s disease, Fragile X syndrome, etc
What does TNRE stand for?
Trinucleotide Repeat Expansion. This expansion can lead to diseases such as Huntington’s disease, and Fragile X syndrome
How are some replication errors able to escape proof reading?
If potential mutations are introduced in the first round of replication through something such as the miscorporation of a base, the 3’ to 5’ exonuclease component of the replisome will come in and remove the wrongly incorporated nucleotides, but some of these may escape this proofreading process. If this misincorpoated nucleotide is not detected and replaced, the sequences change will become a permanent part of the genome. Therefore, in the second round of replication, this wrongly
incorporated base becomes permanent in the DNA sequence and will now be considered a mutation.
What is the mismatch repair system?
This is a method which helps remove the errors that escape the proofreading process in DNA replication. This system increases the accuracy of DNA synthesis by an additional 100-1000 fold on top of the 100-fold by proofreading.
What are some challenges faced by the mismatch repair system?
- Scanning of the genome for a detection of a mismatch can be a difficult process for this system
- Correction of the error in the newly synthesized strand also required a sufficient amount of work
Explain the mismatch repair system in E.coli.
The mismatch repair system in E.coli is known as the MutS/MutL/MutH system.
MutS is a mismatch repair protein that scans the genome and detects mismatches. It has ATPase activity.
MutL is a second protein component of the repair system. It activates the MutH component.
MutH is an endonuclease which creates an incision or neck on one strand of the DNA near the site of mistmatch. Once the incision has occurred, the exonuclease digestion will occur and the displaced single strand will be digested. Then, DNA polymerase III will come in to fill the gaps and ligates the DNA to eliminate the mismatch.
Explain the MutS/MutL/MutH mismatch repair pathway step by step.
The MutS repair protein will identify a mismatch and embrace the mismatch containing DNA which will induce a kink.
Next, the MutS will recruit the MutL and the MutH. The ATPase activity of the MutS will catalyze the hydrolysis of ATP.
MutH, the endonuclease, will create a nick in the DNA near the site of mismatch. Following this, an exonuclease will digest the nicked strand. Finally, the result single strand gap will be filled in by DNA pol III which will eliminate the mismatch.
How does the mismatch repair system known which of the two mismatched nucleotides to replace?
If repair were to occur at random, then the process of removing error would not be very efficient. Therefore, in order to avoid this, E.coli tagging method is used.
This is done through:
The 5’-GATC-3’ sequence is widely distributed in the entire genome. The E.coli enzyme Dam methylate methylates A residues in GATC on both strands of the sequence. When a replication fork passes through the DNA that has been methylated at the GATC sites on both strands (Fully methylated), the resulting daughter DNA duplexes will be heme-methylated (Only parental strand will be methylated). Therefore, the daughter DNA duplexes will be methylated only on the strand that served as the template until the Dam methylase catches up and methylates the newly synthesized strand. Therefore, the newly synthesized strand is marked (by not having a methyl group) and therefore can be the strand that is recognized for repair. The MutH will make incisions only on the unmethylated daughter star.
