Test1: Lect4 Amanda McCullough Flashcards
Nucleotide excision repair (NER):
- Repairs what?
- Repairs what? bulky adduct (Pyrimidine dimers, intra and interstrand crosslinking)
Describe the pathway of nucleotide excision repair pathway in bacteria:
UvrA and UvrB form heterotrimer (2 As, 1 B) ->
UvrC replaces UvrA and makes two incisions, 3’ and 5’ ->
Helicase mediated excision erroneous segment ->
Hifi replication enzymes replace nucleotides ->
Ligase closes the nick
Excision vs incision:
Incision: a cut/nick in the nucleotide strand (takes energy to break the bond)
Excision: Removal of DNA segment (takes energy to pry basepairs apart, often helicase mediated)
What was one of the first clues that repair and replication may be coupled?
Many repair proteins are also part of the transcription complex
Nucleotide excision repair (NER):
- Steps in human:
Recognition and verification (you have to make sure your right before you cut up the genome)
5’ Incision (point of no return)
Repair replication (Minimizes exposure of ssDNA to mutagenic cellular environment, by not completing 2nd incision before replication)
3’ Incision
Excision
Rejoining
How do you verify the damage, (how would a glycosylase do this for example):
base pair flipping. The track along helix til you find something wrong, flipping out the nucleotides into a pocket in the protein which checks the nucleotide.
- Mismatch will check both bases
What does this mean “Nucleotide excision repair is the most “general” of repair pathways”
1: NER doesn’t look for specific mutations, they just look for general errors.
2: This means NER is what helps with new carcinogens. We haven’t evolved any SPECIFIC way to deal with formaldehyde, but NER can just cut out the messed up section
Name a disease caused by a failure in nucleotide excision repair pathway, specifically in Global Genomic Repair::
Xeroderma pigmentosum
Xeroderma pigmentosum (XP): - Symptoms and explanation:
- Symptoms:
Failure of Global Genomic Repair = CANCER
Severe sun sensitivity (cannot repair UV damage)
Skin cancer (from UV damage and other exposures)
30% neurological problems (failure to repair problems in CNS)
Tend to have early death from cancer
How is Xeroderma pigmentosum treated?
It isn’t really treated. You minimize sun exposure, includes using UV filters, and night activities.
Is Xeroderma Pigmentosum dominant or recessive?
Germline or somatic?
Autosomal recessive Germline mutation (inherited it)
What can cells with Xeroderma Pigmentosum not do?
Cannot do repair replication, in NER (process of adding nucleotides while the damaged strand is slowly pealed away)
Cannot do the 5’ initial incision in NER
Different phenotypes of XP made people think that there was different mutations causing it.
How was this tested?
Complementation. Patients cells with a known mutation were tested against patient cells with an unknown mutation. Cells were fused, and given h thymadine a radiolabel. UV exposure, if cells underwent repair radiolabelled T would be added and could be seen later.
If combined cells are equally sensitive as before, they have the same genetic defect.
How many genes are involved in XP?
- How is XP assayed/diagnosed?
at least 8, may be more. And multiple variations may cause it XP.
- How is XP assayed/diagnosed?
1: UV sensitivity assay of patient cells in Vivo
2: Complementation testing
Sequence gene (though made difficult because of reasons listed above)
What are the two types of NER?
Global Genomic Repair: the one we’ve been talking about
Transcription coupled repair
How does transcription coupled repair (NER) differ from global genomic repair (NER)?
1: Instead of having UvrA and UvrB analog find the adduct, the polymerase stalls at it it.
2: Polymerase recruits CSA and CSB which starts the same pathway as global genomic repair (GGR) from here on out.
What pathways are used during transcription transcription coupled repair (TCR)?
BER, NER and translesion synthesis polymerases
Theories as to why transcription coupled repair is important:
- Restart Transcription (allows repair and then start over of transcription)
- Is this just a DNA damage surveillance system?
- Is this a system which measures damage to decide when cell death is necessary?
What happens if you have a defect in CSA or CSB (a key protein in TCR)?
- What, noticeably, is not happening?
Cockayne syndrome
- What, noticeably, is not happening?
No skin cancer
Cockayne syndrome:
- Symptoms
- Why no cancer?
- Genetics:
- Symptoms Cellular UV sensitivity Defective repair of transcribed genes Neuronal issues (unusual myelination Deafness, dwarfism, retinopathy No skin cancer 2 complementation - Why no cancer? TCR is what they are deficient in, this causes cell death when lost but not cancer - Genetics: 1: 2 complementation groups (CSA and CSB) 2: autosomal recessive
cockayne syndrome + XP?
- Symptoms:
- Complementation groups (what is targeted)?
Yes
- Symptoms:
Defective global and transcription coupled repair
- Complementation groups (what is targeted)?
2, XPB, XPD (both helicases, necessary for both global and transcription)
XP Variants still make incision breaks correctly, but have XP, why?
Error in Pol eta, a translesion polymerase which is in charge of error free DNA repair
Where do mutations tend to occur which cause mutator phenotype in pol eta?
in the palm, where it interacts with the DNA
Translesion Polymerases:
- Also called?
- Differ in what way structurally?
- Traits:
- Also called? Slopier copiers - Differ in what way structurally? Looser "finger and thumb", allows replication past adducts - Traits: 1: Poor replication accuracy 2: Favored formation of non watson-crick base pairs 3: can replicate past DNA damage 4: Poor processivity
- How are translesion polymerases recruited?
- Do they fix the damage?
- There are varieties, why?
- How are translesion polymerases recruited?
pol delta stalls, PCNA helps to change affinity, translesion polymerase recruited, replicate past damage, pol delta takes back control. - Do they fix the damage?
No, just bypass it. - There are varieties, why?
They specialize in bypassing specific types of damage. Pol eta bypasses pyramidine dimers (T-T)
How can inhibition of TLS (translesion synthesis) polymerases help patients with cancer?
In some cases, overexpression of these protects cancer cells from chemotherapeutics.
DNA damage response (the bacterial version):
- Define:
- Also called:
- Turned on by:
- Define: turns on 43 genes whenever DNA is damaged - Also called: SOS response - Turned on by: Anything which damages DNA (UV light)
How does the SOS response occur
DNA damaged -> ssDNA -> Binds RecA -> RecA bound to ssDNA cuts LexA -> LexA is the repressor of ~43 genes -> Now they are on instead of off
RecA:
Binds ssDNA to cut lexA (a repressor), involved in SOS.
lexA:
A repressor of many genes, cut by RecA in the SOS response
Mammalian DNA damage response (DDR):
A lot of shiz goes down.
- Chromatin modification
- Translation regulation
- Transcription regulation
- Protein synthesis regulation
- Protein degradation regulation
- Protein modification
- Macromolecular complex formation
Mammalian DNA damage response (DDR):
- Two kinetics:
- Two kinetics:
Fast: protein posttranslational modification
Slow: gene expression and protein synthesis changes
Mammalian DNA damage response (DDR):
- Steps:
Recognize double stranded break break forms, some type of damage ->
Kinases have phosphorylation cascade (H2AX phosphorylation) ->
Cells arrest (give time to fix it) ->
Cannot fix it then cell death
H2AX phosphorylation:
Response to double strand break
Considered a marker of DNA damage.
It is a phosphorylation of the H2AX (Histone 2 AX H2AX, axe made DS break) on chromosomes.
- Spread 2 Mb from site of double strand break
