Nepveu Lectures (Repair Lectures 1-4) Flashcards
(85 cards)
What are the four major threats to the integrity of DNA?
1) Lesions inflicted by endogenous metabolites.
-> Reactive Oxygen Species (ROS).
-> Spontaneous deamination (cytosine, adenine, guanine, and 5’ methyl cytosine)
2) Lesions caused by environmental DNA-damaging events.
-> UV.
-> y-radiations.
-> Chemical mutagens.
3) Replications errors which have escaped the editing process during DNA synthesis.
4) Errors made during DNA repair.
What are responses to DNA damage that are specific to some organisms?
There is conservation of DNA repair mechanisms through evolution.
In microorganisms, there are inducible repair functions.
-> SOS system (counteracts the effects of DNA-damaging agents, this reflects their existence in hostile environmental niches)
-> Adaptive mutagenesis.
Bacteria can also elevate their mutation frequency to adapt to a new environment. This inducibility is absent in mammalian cells, one exception is lymphoid B cells, where somatic hypermuation within immunoglobin genes contributes to antibody maturation.
In higher organisms, there is apoptosis and cellular senescence.
What are the different DNA Repair Pathways?
1) Direct reversal of damage: MGMT, accepts methyl groups from O6-methyl guanine.
2) Base Excision Repair (BER): most DNA lesions from endogenous sources.
-> endogenous DNA lesions arising and repaired in a diploid mammalian cell = intrinsic mutational process as a result of normal metabolism: depurination, oxidation, alkylation of bases are frequent. Then you would do BER to repair this.
3) Nucleotide Excision Repair (NER): bulky lesion causing structural distortions.
4) Mismatch Repair: errors made during replication.
5) Double-strand break repair pathways.
-> Non-homologous end joining (NHEJ).
-> Homology dependent repair (HR).
6) Damage tolerance.
-> Template switching (post-replication repair)
-> Lesion bypass (translesion synthesis)
7) Cell cycle checkpoint control
8) Apoptosis
9) Senescence
What are major sites of cellular reactive oxygen species (ROS)? (Cellular Redox Homeostasis)
ROS (Fenton reaction) do not travel because they are reactive, but can be converted into peroxide, which travels, once in contact with ferrous ions, can be converted to hydroxyl radicals that can cause damage to various molecules, including DNA.
1) Mitochondrial electron transport chain (Mito-ETC).
2) Endoplasmic retculum (ER) system.
3) NADPH oxidase (NOX) complex.
Damaging agent: UV light or polycylic aromatic hydrocarbons.
What is DNA lesion and the repair pathway associated?
DNA Lesion:
- 6-4 photoproduct
- Cyclobutane pyrimidine dimer
- Bulky adduct
Repair pathway:
- Nucleotide excision repair (NER)
Damaging agent: X-rays, Oxygen Radicals, Alkylating agents, spontaneous.
What is DNA lesion and the repair pathway associated?
DNA Lesion:
- Abasic site; oxidized, deaminated, alkylated bases.
- DNA ss break.
Repair pathway:
- Base excision repair (BER).
Damaging agent: Ionizing radiation, Hydroxy urea, UV light, X-rays, Anti-cancer agents.
What is DNA lesion and the repair pathway associated?
DNA Lesion:
DNA ds break.
(can also cause DNA ss break and abasic base site)
Repair pathway:
DS break repair
-> HR
-> NHEJ
Damaging agent: Replication & Recombination errors
What is DNA lesion and the repair pathway associated?
DNA Lesion:
Base mismatches
Insertion
Deletion
Repair pathway:
Mismatch repair (MMR).
There are a few diseases caused by inherited mutations that impair the BER. What would the result of these be?
Embryonic Lethal.
What is the mechanism of BER (Base Excision Repair)? (what are the key players?)
1) BER initiated by removal of modified base by either a monofunctional or bifunctional DNA glycosylase to leave an abasic site (AP).
Pathway A)
Excision:
-> Monofunctional (UDG) (MPG)
Makes AP.
Incision:
5’ APE1 to DNA backbone.
End processing:
PolB or APE1 or PNKP. (depending on specific nature of terminus)
Pathway B)
Excision:
-> Bifunctional (NHT1)(OGG1) (NEIL1)(NEIL2)
Makes AP.
Incision:
3’ AP lyase B-elimination.
NTH1/OGG1 -> APE1 (end-processing)
NEIL1/NEIL2 -> PNPK (end-processing)
Now both pathways have same steps:
Then goes through either short-patch BER/SSBR or long-patch BER/SSBR.
Short patch:
- repair synthesis of the single-nucleotide gap by Pol-B with XRCC1 then DNA ligation by LIG3 and then repair is done.
Long patch:
- repair synthesis of 2-13 nucleotide gap by Pol-B and/or Pol delta or epsilon aided by PINA and RFC.
-resulting 5’ flap removed by FEN1.
-final ligation by LIG1.
In BER, what happens if single-strand breaks occur by other means?
They can contain simultaneous 3’ and 5’ obstructive termini.
-> PARP1 recognizes these breaks and start performing ADP-ribosylation, & end processing takes place, uses TDP1 and APTX.
What is the connection between base excision repair and transcription?
- Some TFs function as accessory factors in BER.
- They stimulate enzymatic activities of BER enzymes.
- DNA de-methylation is performed by BER.
- MPG interacts with RNA pol II through direct interaction with the elongation complex. Active transcription elongation promotes efficient MPG-directed repair.
- Some BER enzymes can recruit a TF close to a promoter.
What are the two pathways of mammalian nucleotide excision repair?
1) GC-NER: global genome excision repair.
2) TC-NER: transcription-coupled nucleotide excision repair.
- In TC-NER, Single Strand Break protein travels with Pol II and recruits DNA repair proteins when RNA Pol II stalls at site of DNA damage.
What is the mechanism of Nucleotide Excision Repair (NER)? What are the key factors?
(1) DNA Damage Recognition
- Helix distortion damage takes place.
- There are two pathways, GC-NER and TC-NER.
Pathway (1) GC-NER:
- recognized by XPC complex ((DDB1, DDB2) known as XPE), facilitate recognition of lesions.
- XPC complex recruits TFIIH to the repair site.
Pathway (2) TC-NER:
- recognized by the stalling at RNA at Pol II, facilitated by CSB, CSA, XAB2.
- CSA recruits TFIIH to the repair site.
(2) DNA Unwinding.
- XPD and XPB subunits of TFIIH are DNA helicases that unwind the DNA in the immediate vaccinity of the lesion.
- RPA and XPA bind to keep the DNA strands apart.
(3) For dual incision:
- XPA recruits XPF-ERCC1 endonuclease to incise the damaged DNA strand 5’ to the lesion while XPG incises 3’ to it.
(4) Repair synthesis:
- performed by DNA polymerase delta and kappa or epsilon with help of accessory proteins RFC, PCNA, RPA.
(5) DNA Ligation:
- LIG1 or LIG3a-XRCC1
In terms of TC-NER, what is a feature of CS that has to do with this pathway?
*** normal human cells preferentially repair the transcribed strand of active RNA polymerase II transcribed genes. Cells from patients affected by Cockayne Syndrome (CS) do not show this preferential repair. Two complementation groups have been identified for CS: CS-A and CS-B.
What are human diseases caused by defects in NER?
1) Xeroderma Pigmentosum (XP)
- characterized by clinical photosensitivity
- XP-A and XP-G complementation groups identified.
2) Cockayne’s syndrome (CS)
- sun-sensitive, distinctive array of congenital neurological and skeletal abnormalities.
- CSA & CSB.
3) Trichothiodystrophy (TTD)
- share many symptoms with CS, definitive symptoms is brittle hair & nails.
- mutations in at least 3 TFIIH genes are implicated in TTD: XPD, XPB, TTDN1
** fibroblasts derived from patients with these UV sensitive syndromes have been instrumental in identifying proteins involved in NER pathway.
What is the mechanism of Mismatch Repair in E.coli? What initiates this mechanism?
It is initiated by mistakes during replication due to slippage of DNA replication machinery along the DNA template in regions of simple repeat sequences.
(1) MutS
(2) MutL & MutH bind + ATP
This leads to the introduction of distant strand break in the newly synthesized DNA strand.
(3) MutH recognizes hemimethylated d(GATC) sequence and cleaves the strand.
(4) UvrD protein, DNA helicase, unwinds the DNA from the nick, the displaced DNA strand is degraded by exonuclease + ssb (protecting remaining strand).
(5) gap filling by DNA Pol II & joining by DNA ligase completes the repair process.
What is the mechanism of mismatch repair (MMR) in humans?
(1)
- Recognition & binding of mismatch by MSH2-MSH6 or MSH2-MSH3.
- Recruits MLH1-PMS2 complex (form ternary complex).
(2)
- Excision by 5’ to 3’ exonuclease EXO1. RPA binds to protect SS-DNA during excision.
(3)
- repair synthesis is accurately performed by Pol Delta.
(4) Ligation of the remaining nicks after synthesis is by LIG1.
What is implicated in determining the direction of mismatch repair in mammalian cells?
- PCNA.
In Human MMR, what happens if you need to do 3’ directed MMR?
- endonuclease function of PMS2 is activated by ‘ nick, stimulated by RFC & ATP, to make the 5’ nick, the excision can follow by Exo1.
What is Gene Conversion? What is co-conversion?
- Gene conversion happens during DNA repair when two similar but slightly different DNA sequences exchange genetic information.
- If there is a mismatch (base pairing error) between the two DNA strands in a region called heteroduplex, the cell’s MMR system comes to fix it.
- Since each heteroduplex region has 2 strands, there are two heteroduplex regions per recombination event, there are four possible ways mismatches can be repaired.
- Co-conversion: if there are two mismatched sites in the heteroduplex DNA that are close together, the mismatch repair system might fix both sites during same repair event.
- Gene conversion can happen with or without recombination. With, the sequence on either side of the heteroduplex swap places between the two DNA moleciles. Without, only heteroduplex regions is affected, regions on either side stay the same.
Homeologous Recombination
- Recombination between two DNA molecules that diverge at many positions is inhibited by MMR pathway.
- The capacity to perform homeologous recombination is believed to help pathogenic bacteria to acquire new traits.
How is a single oxidative DNA lesion repaired vs a clustered oxidative DNA lesion? What causes them?
- They are caused by Ionizing Radiation.
- Single is repaired by BER.
- Clustered is repaired by BER, NHEJ, and NER.
These are all DNA repair mechanisms.
What are 3 ways that double-stranded breaks are produced? (how are each repaired?)
(1)
DBS’s generated when the two complementary strands of DNA are broken simultaneously at sites that are sufficiently close to one another, that base-paring and chromatin structure are insufficient to keep two DNA ends juxtaposed.
Repaired by HDR & NHEJ.
(2)
Replication through a region with SSB can cause a DSB (“double-strand end”-DSE)
Repaired by HDR.
(3)
Repair of damaged bases that are close to one another can generate “Secondary DSBs.”