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BMB 400 -chapter 10&11
Molecular Biology of the Gene
65
Biochemistry
Undergraduate 3
10/02/2016

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Term
What is the difference between a transversion and a transition?
Definition
transversion: pyrimidine - pyrimidine , purine - purine mix ups
transitions: pyrimidines- purines mixups
Term
What is a DNA micro satellite?
Definition
a "hotspot" for a mutattion to occur: if there are long repeating sequences of CA
Term
If a mutation isn't going to be permanent, when does it have to be found by?
Definition
before the second round of replication
Term
how does mismatch repair affect mutation rate in E. coli cells?
Definition
100-1000 times
Term
What is the mismatch repair system in bacteria ? How does it work ?
Definition
MutS recognizes dimers from the distortion in the DNA backbone. Must binds to it, and it induces a conformational change in the MutS. MutS has ATPase activity that is required for mismatch repair. MutS recruits MutLwhich recruits MutH. MutH nicks one strand near the mismatch. It then eats from the nick to past the mismatch. Ligase then fills it in.
Term
what does MutS use as a cofactor?
Definition
atp
Term
How does the mismatch repair system recognize the newly synthesized DNA?
Definition
MutH can only recognize the "hemimethylated" DNA, which is the newly synthesized DNA since Dam methyltransferase hasn't had a chance to catch up to DNA polymerase and methylate it. Then MutH nicks it
Term
What is the Ames Test?
Definition
it's a test that tests mutagens on bacteria to see if they will grow even if they are under unfavorable conditions. (see if chemical will revert growth conditions)
Term
How do bases get deaminated? What is the result ?
Definition
When water is added to the bases, cytosine loses it amine group, guanine loses its purine group (depurination), when 5-methylcytosine is added to water, then it loses it amine group.
Term
Why does DNA have thymine instead of uracil?
Definition
If DNA had uracil instead of thymine, then the deamination of cytosine would yield a base that would be natural and the repair systems wouldn't work.
Term
how does gamma radiation and x rays affect DNA?
Definition
double strand breaks
Term
What does photo reactivation fix?
Definition
pyrimidine dimers
Term
Which enzymes fix double stranded breaks in E. coli?
Definition
RecA and RecBCD
Term
What is DNA photolysase?
Definition
uses energy from light to fix thymine dimers
Term
Sometimes DNA gets methylated from UV radiation. How does it get fixed?
Definition
methyl transferase will come and remove the methyl group from the base.
Term
Give an example of coupling reaction and mutagenic base repair
Definition
photoreactivation
Term
Which enzyme is involved in base excision repair?
Definition
DNA glycosylase.....cuts out bases that have flipped to the outside on minor groove by hydrolyzing glycosidic bond
Term
Why is oxoG so mutagenic?
Definition
It can pair with both adenine and cytosine
Term
if oxoG pairs with A during replication, what mutation has occurred?
Definition
It was a G:C to a A:T....so a transversion
Term
how do you make oxoG
Definition
oxidation of guanine
Term
what does a fail-safe glycosylase do?
Definition
it identifies the replication product of oxoG (daughter strand) and removes the A base pair, replaces it with C
Term
How does nucleotide excision work?
Definition
Recognizes distortions to the shape of double helix (thymine dimmer or by the presence of a bulky chemical adduct). Cuts out the section and uses DNApolymerases to fill in the gap.
Term
True or false: RNA polymerase is a damage sensitive protein
Definition
YES TRUE. because mechanisms of repair are more efficient when they are on the template strand for transcription.
Term
What does TFIIH do in transcription-coupled repair?
Definition
it acts as a helicase
Term
How does nucleotide excision work?
Definition
Recognizes distortions to the shape of double helix (thymine dimmer or by the presence of a bulky chemical adduct). Cuts out the section and uses DNApolymerases to fill in the gap.
Term
True or false: RNA polymerase is a damage sensitive protein
Definition
YES TRUE. because mechanisms of repair are more efficient when they are on the template strand for transcription.
Term
What does TFIIH do in transcription-coupled repair?
Definition
it acts as a helicase
Term
what is the mechanism for double strand break repair?
Definition
there are two ways: one uses homologous recombination with the non damaged sister strand of DNA as the template---introduces no mutations. The other way is to use NHEJ, which sticks two ends of chromosomes together
Term
why do eukaryotes use NHEJ rather than prokaryotes?
Definition
we have more introns to spare
Term
In eukaryotes, which type of polymerase is able to carry out translesion repair?
Definition
Y family---it is able to incorporate nucleotides from template and without a template (SOS response)
Term
in e. coli, what is the name of the polymerase that can do translesion repair?
Definition
DNA Pol IV and V
Term
Which type of translesion polymerase is better for fixing thymine dimers? Why?
Definition
Pol Eta, has a more open pocket
Term
What is the difference between the polymerase switching and the gap filling model ?
Definition
polymerase switching is the when the replicative polymerase comes to the mutation site, falls off, and the translesion polymerase hops on. the gap filling model is when the replicative polymerase just jumps ahead
Term
Does the Holliday model start with a single or double strand break?
Definition
single strand
Term
Why does the "Double Strand Break Repair" introduce ssDNA 3' ends?
Definition
so that both can invade the other whole chromosome
Term
What is the RecBCD pathway?
Definition
a method of DSB repair
Term
Which enzyme involed in the RecBCD pathway has the ability to be a helices and a nuclease?
Definition
RecBCD
Term
which sequence elements control RecBCD?
Definition
chi sites
Term
What is the difference between RecB and RecD subunits?
Definition
they both are helicases and use ATP hydrolysis, however Rec B has a 3 to 5 helices and has a multimulctional nuclease domain
Term
Which subunit of RecBCD leads the complex ?
Definition
D
Term
What does the C subunit of RecBCD do?
Definition
recognizes CHI sites
Term
Role of RecA?
Definition
strand invasion and pairing homologous DNAs
Term
Which enzyme breaks double stranded broken DNA into single strands to prepare for homologous recombination??
Definition
RecBCD
Term
how does the RecBCD pathway work?
Definition
RecBCD is gliding along the DSB and is cutting both strands into little pieces. Then it encounters the X sequence, and stops for moment. Then it continues on at the rate half as fast as it was moving before and then it starts chopping more on theater strand.
Term
In the Holliday model, how many nicks in how many DNA strands initiates the homologous recombination?
Definition
2 nicks, in two identical DNA strands but in two different DNA duplexes
Term
how many nucleotides long are X sites?
Definition
8
Term
There is 1 RecA for every _________ nucleotides of sand.
Definition
3
Term
What are the two binding sites of RecA?
Definition
Primary: binding the ssDNA
Secondary: binding the dsDNA, while scanning for complementary sequence. Doesn’t need a specific sequence to bind
Term
In the DSB model, When the ssDNA actually invade ?
Definition
when a complementary sequence is found. RecA catalyzes the H bond between the two sequences
Term
Which model of DNA homologous recombination uses a Polymerase and ligase?
Definition
---NOT holliday model, so the DSB method
Term
What does the RuvAB complex do?
Definition
initiates the branch migration!!
Term
What does RuvA do? What is it?
Definition
it is a DNA binding protein that recognizes the crossover sites regardless of specific sequence
Term
How many RuvB molecules does RuvA recruit to the Holliday site? What is RuvB?
Definition
it is an ATPase, that catalyzes the actual migration
Term
What is special about the resolving of the DSB model ?
Definition
depending on how it is cut, (by RuvC) two different products can be formed. either non-crossover (identical to parents) or splice products
Term
How many sites are there for cutting per Holliday junction?
Definition
2: each one has a crossover and parental strand[image]
Term
What happens if the Holliday junctions are cleaved in the same way?
Definition
then patch recombination results (like parent, no crossing over occurs)
Term
What happens if the Holliday junction are cleaved in different ways?
Definition
Then splice recombination products results
Term
What is SPO11?
Definition
introduces double-strand breaks, cut-sites are located most commonly in chromosomal regions that are not tightly packed with nucleosomes such as promoters (hotspots for recombination)
Term
How does SPO11 cut the DNA?
Definition
Tyrosine side chain attacks the phosphate backbone to cut DNA, and generate covalent complex between protein and DNA.
Term
WHat are the benefits of the eukaryotic covalent complex formation in homologous recombination?
Definition
The bond energy that it takes to break the chromosme into two is stored within the protein-DNA linkage. So that same energy is reharnessed when the cleavage reaction is reversed.
Term
What is the MRX enzyme complex?
Definition
The RecBCD of eukaryotes
Term
What is Rad51 and Dmc1?
Definition
bacterial RecA homolog in eukaryote and play a role for meiotic recombination
Term
When are Rad51 and Dmc1 expressed?
Definition
Rad51 expresses both mitotically and meiotically, but Dmc1 is expressed in only meiosis
Term
What's the difference between SPO11 and HO?
Definition
Spo11 keeps DNA covalently linked, HO hydrolyzes the DNA
Term
WHat is HO?
Definition
endonuclease involved in homolgous recombination in yeast
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