lecture 17 Flashcards
(23 cards)
how is mutation rate measured?
what is the reason why genes can be mutation hotspots?
by phenotype or by counting frequency per base pair
–mutations at phenotypic level are most common
-large size
what was the experiment that fluctuation test?
what were the two mutation hypotheses, how did they apply to this experiment?
tested the nature of bacterial mutations that produced resistance to bacteriophage infection— protects E coli from lysis
1– random mutation—> which was correct, saying that they occur at random
-random cultures would develop mutations at different times, lots of variation in mutation between cultures
2–adaptive mutation–> environmental changes trigger mutation
-cultures exposed at the same time to a trigger would respond the same way, little variation of mutation between cultures
what is the difference between germ-line and somatic mutations?
germ-line: can be passed on from one generation to the next
somatic: can be passed on via mitotic division, but only direct descendants of the mutated cell carry the gene
what are examples of coding sequence mutations?
coding :
synonymous– change in 3rd base, no change
missense– changes aa
nonsense– creates stop codon, stops translation
frameshift: insertion that shifts the aa frame, loss of protein function
what are base pair substitution mutations?
transition–purine replaces with another purine, pyrm replaces with another prym
-most common
transversion–> purine is replaced by pymidine
what are regulatory mutations?
where do they occur?
regulatory: alters wild type protein product
at promoter— changes timing or amount of transcription
noncoding regions:
promotoer, introns, 5 UTR, 3 UTR
what are promoter mutations? results?
alter sequence of nucleotides and interfere with efficient transcription
B globin is example
results: reduced amount of transcript & reduced protein , could completely eliminate transcription
what are splicing mutations? results?
GT occurs at 5 splice site of introns normally
AG at 3 end of exon in B globin
- mutations of these areas or nearby nucleotides within the intron can mess up splicing
what are cryptic splice sites? result?
base pair mutations can cause new splice sites that replace or compete with authentic splice sites during pre mRNA processing
-can leave additional nt in mature mRNA
what is polyadenylation mutations? results?
processing of 3’ end of eukaryotic mRNA is inited by 5’ AAU – AAA 3’
leads to abnormal mRNA and reduction of functional protein
what are types of spontaneous damage to individual nucleotides?
** DNA replication may need to occur before they are preserved as mutations in DNA**
-depurination: loss of one of the purines (A or G) from a nucleotide via breaking covalent bond at 1’ carbon that links the sugar to the base, leading to—> apurinic site
- purines are most commonly lost & most are replaced
-deamination: loss of Nh2 amino group from the nucleotide base
-deamination of Cytosine, NH2 is replaced by O–> forming uracil
-deamination of 5MeC–> makes Thymine (mutation hotspots)
Oxidative damage: ROS species, wrong base pairing
mutagens are used to create ____ mutations?
examples include?
induced
nucleotide base analogs: compound that has similar structure to DNA bases & can be worked into DNA
-BU can mess with A and G base pairing
deaminating agents:
-nitrous acid removes NH2 group, can switch from A/T to G/C
alkylating agents:
-add bulky side groups CH3 and ethyl groups
-EMS
hydroxylating agents:
-adding OH–> mis pairing
Intercalating agents:
-squeeze their way in-between DNA base pairs and mess up the duplex
-lead to breakage of phosphodiester bond on 1 strand
-causes frameshift
what is the AMES test?
rat of new mutations by identifying the rate of revision mutations measures the ability for bacteria to restore their own HIS
results: by counting number of growing colonies on each plate & comparing wit another on control plates
what is photo reactive repair?
– in bacteria
uses photolyase to bind to UV induced photo produce
how does base excision repair (BER) work?
5 steps
-presence of incorrect base initiates this
-error free
1: DNA N glycosylase recognizes base pair error
2:DNA N glycosylase removes the base sugar bond
3: AP endonuclease cuts sugar phosphate BB at 5’ of the AP site—> giving nick
4: dRpase removes stretch of DNA
5: nick translation–> DNA pol recognize nick & initiates removal of DNA nt & replaces them
6: ligase seals the backbone
what is the purpose of methylation in DNA mismatch repair?
how does mismatch repair work ?
-identifies which strand based on methylation pattern post replication & is coupled to replication
-parental is methylated on the A after replication , daughter is hemi-methylated
1: MUTs searches daughter strands to see if any errors in base pairing, recruits MutH and MutL
2:MutS: binds to site of mismatch & forms complex with MutH
3: MutH breaks the phosphodiester bond on 5’ & exonuclease gets rid of the mis pair and sequences beyond the mis pair
4: DNA pol fills gaps & DNA ligase
5: methylate daughter strand
** no MutH in eukaryotes***
what are the steps of nucleotide excision repair (NER)?
-error free to repair UV induced damage due to bulky mutations not being a sustrate for DNA glycoslase
1: detects distortions in base pairs via XPC complex
2: TF11H (XPB and XPD) unwind DNA around lesion to create open repair bubble
3: XPG endonuclease cuts the damaged DNA strand 4-8 nt downstream of the lesion and 15-24 nt upstream of lesion
4: DNA pol fill missing nt, using undamaged as the template and sliding clamp PCNA makes sure its right
- Replication protein (RPA) stabilizes the single stranded region
5: ligase 1 fills the nick
how does translesion synthesis work?
1: Pol 3 stalls at site of damage
2: replaced by bypass pol V, which adds random non pairing nt, only synthesize short parts
- no proofreading
3: bypass pol 5 then falls off, then pol 3 continues synthesis
- error prone
how does non homologous end joining work?
- for double strand break repair , ERROR PRONE because removes nt that cannot be replaced
1: KU80-KU70-PK protein binds broken ends of DNA
2: trims free ends of each side, leaving blunt ends and loss of nt
3: DNA ligase ligates bound ends to reform the duplex
how does synthesis dependendant strand (homologous directed repair) annealing work?
– for Double strand break repair, error free
1: trim one end of the broken strand
RAD51 attaches to unbroken strand
2: RAD51 allows for strand invasion
3: strand invasion displaces on strand of the intact duplex & creates D loop–> results in holiday junction
4: replication within the D loop synthesizes new DNA from template strands
5: unwind from template and anneal back to normal
6: ligate of the gaps via ligase
how are errors corrected with during DNA synthesis?
where does pol 3 and pol 1 hold the DNA?
stops and reverse the replication to remove the incorrect nucleotide because the template and daughter are not able to properly H bond
-3—5 exonuclease activity removes the incorrect base pair, synthesis is resumed
thumb and fingers hold the template + daughter strand in the palm
-
what is the SOS response?
uses translesion synthesis in bacteria
RecA is pos regulation
LexA is a negative regulator
uses Pol 4 and 5 that use non coding bases as template
how do nonsense mutations look on northern and western blot?
RNA will be the same, but the protein can be smaller
higher on N blot and W blot
