Mutation and DNA Repair Flashcards
What is a mutation?
- 2 ways they can result
- How does it cause a problem?
Mutation: An inheritable change in DNA
-Results from damage or chemical changes to DNA bases - can occur spontaneously or be induced.
- Mutations change bases’ binding specificity (i.e. so A binds to G)
- next round of replication introduces a mutation (CG pairing that wasn’t present before)
- mutations are fixed in the genome of the cell
How often do mutations occur?
- In E. coli
- For a double mutant - spontaneous mutation frequency
- In a broth culture of E.coli, there will be mutants.
- to isolate a lacZ- mutant, need to screen approx. 10^7 cells
- spontaneous mutation frequency = 10^-7
- To find a lacZ- LacA- double mutant, would need to screen approximately 10^14 cells
Spontaneous Mutation Frequency among different species
- In humans
- In immunoglobulin genes
- Frequency & exposure to mutagens
- Mutagen definition
- Varies among species
- In humans; is approximately 10^-5
- Some genes have different spontaneous mutation rates
e. g. Immunoglobulin genes (antibody genes) = 10^-3- Hypermutable because you need diversity in antibodies for protection against virus, bacteria etc.
- The frequency can be increased by increasing exposure to DNA damaging agents (called MUTAGENS)
- Mutagens = chemical or physical agents that cause chemical changes in DNA bases)
How do we know mutations are spontaneous?
-experiment involving E. coli
- E.coli onto streptomycin agar and incubated -> if replicates are made all plates will have the same pattern/distribution of growth on the plates
- demonstrates that mutations pre-existed in the culture and were not caused by the exposure to streptomycin
*defines the nature of mutations -> random
Effect of mutagen/mutation
-depends on 2 factors
Depends on;
- Type of mutation
- Location of mutation
*Don’t necessarily see an effect of the mutation
2 Types of mutations
- Missense: Change of one amino acid for another
2. Nonsense: Change of one amino acid codon with a stop codon
Nature of codons & effect of mutation
-Which of the two types is worse?
- Are approximately 4 codons for every a.a.
- Changing first or second base in most cases will change the a.a.
- if a stop codon is introduced early on, it will stop translation and the protein won’t be made = MAJOR EFFECT
- Nonsense will have a much greater effect as the protein won’t get made!
Missense mutation Effect
-conserved region -> what is it and why is it important when thinking about mutations?
- The effect of missense mutation depends on where the mutation is
- protein sequence tends to be different between species, even though the protein may be the same
- Conserved region = small portion where all proteins have same a.a. sequence
- this region is therefore critical to function of that protein
- rest of protein may be involved in other less critical functions
- mutation in conserved region likely to be more disastrous than in other regions
Effects of mutations within cells
- only 2.8% of human genome encodes proteins
- 28% is transcribed (e.g. tRNA, rRNA & regulatory RNAs)
- Approx. 70% of our genome don’t encode for anything - therefore a very small amount of mutation would affect our proteins
- Being diploid also protects us - effect of a mutation can be masked by copy on sister chromosome
Frameshift mutations
- what are they
- why are they significant
-what can induce a frameshift mutation?
- are the insertion or deletion of 1 or more bases in mRNA that changes the reading frame and therefore completely changes the protein
- have a profound effect on the gene
- Frameshift mutagens can induce this change
What else can also cause mutations?
- what this does
- likely effect in humans
- but what it can do to a gene that has significant effects on expression
- DNA arrangements can also cause mutations
- are quite common b/w DNA molecules that are quite similar - they can undergo cutting and joining (i.e. recombination)
-if small copy of a DNA sequence is similar but in reverse orientation, it may cause pairing
-net result is the inversion of the sequence between the reacting sites
*as human genomes have small amount of coding genes, it’s unlikely to effect a protein
BUT, inversions can move a gene from one place to another & the position of a gene on the chromosome can have significant effects on expression
How location on a chromosome effects expression of a gene
- length of telomere
- e.g. of eye colour in Drosophilia
Telomeres induce the formation of heterochromatin (as are the DNA around the centromeres (tightly packed DNA)
- genes in heterochromatin are not expressed (even when the right activators are present) * most of chromosome exists as euchromatin (which is not as tightly packaged)
- Length of telomere, and hence length of the heterochromatin can very from cell to cell -> gene may be expressed in some cells but not in others
- e.g. eye colour in Drosophilia (inversion = patchy red colour compared to red colour in wildtype = position effect variegation)
What inversions do (in general)
-Inversions exert an effect by moving genes into or out of regions of heterochromatin
Result of directly repeated sequence in DNA
- Result = DNA that lies between the sequence can be deleted
- deletion can range from 100s of kB
Recombination & chromosome number
-effects on humans and plants
- Recombination can fuse chromosomes together so that 1 of the daughter cells at cell division receive an extra copy of part or all of a chromosome
- trisomy
- occurs in plants a lot, but plants can deal with it well
- in humans, trisomies can have a very significant and negative outcome (i.e. Down syndrome)
What are rearrangements in the chromosome mediated by? (2)
- General recombination proteins (act on homologous sequences - i.e. crossing over at meiosis)
- Mobile genetic elements & their enzymes: are designed to move around and undergo recombination reactions
- they play a critical role in evolution of our genome
- also provide homologous sequence for inversion and deletion as when they move, they leave a copy behind
Importance of Mutation
- Mutation is v. important for species evolution
- leads to the formation of new genes in the population, which is essential to adaptation of the species
-e.g. of Biston moth (black and white variants, black ones become more predominant after industrial revolution and trees become black with ash)
DNA Damage - what it leads to
- DNA repair systems (2)
- what they each do
DNA Damage -> mutation
- too much mutation is a bad thing - Not all damage leads to mutation -> some can be reversed by DNA REPAIR SYSTEMS (is the major repair process) - is also an error prone DNA repair system that introduces mutations (operates as a last resort on DNA that would otherwise die)
Error Free DNA Repair System
- How it works
- important enzyme
- Is a nucleotide excision Repair (NER)
- damage introduced into cell by damaging base in DNA - this causes a kink/irregularity in the DNA
- enzyme (Glycosylase) cuts bond between base and sugar (which is a Glycosylic bond)
- The damaged base is released
AP endonuclease -> role in Error Free DNA repair system
- backbone is cut on the 5’ side of the AP site (where there is base missing) by an AP endonuclease (causes a gap on a single strand)
- DNA polymerase comes along to do its thing (degrades front and synthesises behind it & disassociates after approximately 15 bp)
- DNA ligase then comes and seals the gap
- DNA polymerase comes along to do its thing (degrades front and synthesises behind it & disassociates after approximately 15 bp)
Specificity of NER
- Recognises a very wide spectrum of damage
- NER removes damage induced by sunlight, those that don’t have it are very sensitive to it
*in somatic cells, on average, have approximately 1000-10000 DNA repair events per day
Mismatch repair system
- what it does & where mainly involved
- 2 main proteins
-effect of mutated strains of E.coli that dont have these proteins
- Is responsible for removing mismatched bases during replication (that occur in replication fork)
- Mut S & Mu L proteins = prime ones in recognition of mismatchs and initiating repair
- are both a part of replisome
-strains of E. coli that are deficient in these proteins have a mutated phenotype & a spontaneous level of mutation that is 1000 fold higher
Process of Mismatch repair system
- Mut S & L recognises mismatched base, recruites uvrABCD complex
- uvrABCD complex makes a cut on either side of the newly synthesised strand with the wrong base
- results in a gap = replaced by DNA polymerase (DNA ligase seals them together)
How The mismatch repair system knows which base is the wrong one
-hemimethylated DNA
- After DNA is synthesised, modifications are made to the DNA (i.e. methylation)
- BUT there is an interval between synthesis and methylation -> is how mismatch repair system knows which strand is ‘right’
- parental strand will be methylated whereas the newly synthesised strand won’t be
- DNA adenine methylase is enzyme that adds methyl groups to GATC sequences
- Hemimethylated DNA = region behind replicating fork
-mismatch repair system in Bacteria (What it’s called)
- In bacteria, mismatch repair system is called the methylated instructed mismatch repair (MIMR)
- mutS dimer binds to mismatch - mutL dimer then binds to mutS
- mutS then translocates to GATC, which causes DNA to loop
How to check which base is right in Eukaryotes
- is different because DNA in eukaryotes is much more methylated
- instead; uses the end of DNA strand: newly synthesised strand will have the end quite close (parental strand will have its end quite far away)
- is how MMR can recognise which strand to fix
*if genes for repair systems inactivated -> tumors develop
Mismatches during recombination
- Mismatches can also occur in recombination (crossing over at meiosis between sister chromosomes)
- although they are homologous, there are still some differences between them
- DNA that has undergone crossing over = heteroduplex DNA
- mismatches will be repaired by MMRs, unless TOO different - then the process will be aborted.
Error Prone Repair System - what it is
- when it is used
- process of initiating it
- Non coding lesions (NCL) are a lethal event for that replicon, as DNA can’t be synthesised and is therefore stopped
- to overcome this problem, use EPR
- is based on an error prone DNA polymerase
- to overcome this problem, use EPR
- DNA polymerase attempts to incorporate a base into growing DNA strand
- no nucleotides in the NCL makes DNApoly think it’s a mistake and removes it
- keeps going through cycle of trying to incorporate bases that don’t H bond and removing it
- this process accumulates dNMP, which induce the expression of error prone DNA polymerase
- no nucleotides in the NCL makes DNApoly think it’s a mistake and removes it
Error Prone Repair
- Error Prone DNA poly undergo translesion synthesis
- EP DNA poly replaces DNA polymerase -> inserts bases at random so DNA synthesis can continue beyond lesion
- it is a mutation; probably won’t insert the right base, but most of the time it won’t have a major phenotypic change (due to small % of protein coding regions)
- better to have a mutation, than no protein being synthesised (as latter is way worse)
*is why it is also called a SOS repair system
Tendencies to be error prone in Eukaryotic polymerases
-How Error prone DNA polymerases are regulated
-out of the 5 DNA polymerases in eukaryotes, 3 are error prone
-problem = also causes mutation in undamaged DNA
Therefore need to regulate the expression of these
-when nucleotide monophosphates accumulate they act on recA (non protease) protein, which activates to become recA* (RecA star) (which is a protease)
-LexA normally represses the SOS genes
-but after a DNA damaging event, RecA is activated (to RecA*) which cleaves the LexA and allows induction of SOS genes
