DNA Repair Flashcards
Types of DNA damage
Change of base (spont. base loss, deamination, UV induced thymine dimers, base alkylation and base oxidation by ROS)
Change in DNA Structure
(Bulges in helix caused by insertions, deletions, bulky chem adducts, rep errors, cross links; strand breaks, stalled replication forks)
An example of deamination w/ change in base.
cytosine deamination goes to uracial (CG to TA in structure)
Base alkylation example for point muation
GC (to O6-meG:T) to AT
Xeroderma pigmentosum
increased sensitivity to sunlight. Skin neoplasms and Neuro degen later. Genetic defect inactivating nucleotide excision repair (Global genome NER). Pyrimidine (thymine) dimers are passed over by repair mech, meaning increased probability of mutation/skin cancer risk.
DNA structure changes interfering w/ replication and transcription
Double strand breaks
Interstrand (2 sep strands) crosslink
Intrastrand crosslink
BPDE (Guanosine BPDE, a base adduct ex, causes insertion of A opp G by DNA pol)
Direct reversal of damaged bases
- Repair single strand break w/ DNA ligase
- Reverse UV damaged bases (T-T-T-C dimers) using photlyase
- Reverse base alkylation of O6-meG using MGMT (O6-meG methyltransferase)
Base excision repair (BER)
repairs base damages that do not distort DNA
-uses base specific glycosylases to remove damaged base
Nucleotide Excision Repair (NER)
repairs base damages that distort DNA
-removes an oligonucleotide that contains the damaged base
Mismatch repair
removes misincorporated nucleotides during DNA replication
-can tell difference b/t template and new strand
MGMT
coserved direct reversal example of DNA repair
Removes methyl group from O of Guanine on C6
Lesions corrected by Base Excision Repair
deamination dealkylation oxidation spontaneous base loss (doesn't distort DNA)
Mechanism of Base Excision Repair
- Modified base recognized by spec. DNA glycosylase, which hydrolyzes the N-glycosidic bond (cleaves base) yielding an AP (abasic) site
- AP-site specific endonuclease (like APE1) cleaves sugar phosphate backbone in 5’ direction
- A second endonuclease cleaves 3’ to AP site, removing deoxyribose phophate
- DNA Pol fills gap and DNA ligase seals nick.
Lesions corrected by NER
thymine dimers, crosslinking, bulky adducts, nucleotide insertion or deletion.
-distort DNA
Steps of NER
- Recognition and binding of damaged site by multi-protein complex (2 possibilities)
- Local unwinding with helicases of TFIIH to form ~25 base bubble.
- 2 endonucleases, double incision, remove ~30 base oligonucleotide w/ lesion.
- Gap filled w/ DNA Pol
- Nick fixed with DNA ligase.
How does NER machinery recognize damage?
- Global genome NER (recognizes damage anywhere in genome)
Defect–>cancer; XD - Transcription-Coupled NER (recognizes damage in transcribed regions)
Defect–>CNS disorder like Cockayne Syndrome (CS)
Cockayne syndrome
Defect in transcription coupled NER.
- Sun hypersensitivity
- Premature aging (progeria)
- Neuro degen
- Impaired development
Mismatch Repair (MMR) mechanism/what repairs?
Repairs mistakes by DNA Pol in replication.
1. Mismatch on daughter strand recognized by hMSH and hMLH/(PMS)
(in pic hMSH2 and hMLH1)
2. Endonuclease cleaves phosphodiester backbone
3. DNA helicase unwinds double helix [order?]
4. Exonuclease removes mismatch-containing new nucleotide strand
5. DNA pol fills gaps, DNA ligase seals nicks
How does MMR recognize which strand is the right strand to repair?
New lagging strand has transient 5’ DNA ends of discontinuous Okazaki fragments.
New leading strand has transient ribonucleotides (1 rNMP/1250 dNMP), processed into nicks by RNase H2.
MMR gene mutation causes ____
HNPCC (hereditary non-polyposis colorectal cancer)
- 5% of all colon cancer
- one of most commonly inherited cancer-suscep.
What can be utilized when damage to DNA is too great to be fixed?
Cell can go to last resort of employing “bypass” or “error prone” DNA polymerases which have loosened specificity. Allows replication to continue through damaged template strand.
Error rate is 100-100,00 fold higher than normal DNA pol.
What does bypass DNA Pol lack?
3’ to 5’ exonuclease proofreading activity.
Types of bypass polymerases in translesion synthesis
Poli, Poln (greek letters)
Poln: extends synthesis over lesion
Poli: extends synthesis a little past lesion
Both attach to replication complex at stall.
Both fall off after lesion
NHEJ
non-homologous end joining
Ku proteins bind end of double strand break, recruit kinases (DNA-PKcs) for end joining
W/ kinase activation, short strand binds with complimentary other strand
DNA pol fills gaps
Free ends ligated, unpaired removed (can cause deletions and insertions)
HR
Homologous recombination repairs double stranded breaks
-3’ ends digested by exonucleases
-Invade duplex of intact DNA homolog
-Holliday intermediates form(rep by DNA pol)
-cleavage of phoshpdiester bonds
-DNA ligase seals nicks
ACCURATE
What is activated by single strand breaks? What is added?
PARP: Poly(ADP-ribose) polymerase Adds poly(ADP-ribose) chains to proteins.
PARP’s role in SSB repair
- amplifies damage signal
- Focal enrichment of repair proteins
- Change in local chromatin structure.
What does DNA damage checkpoint do?
Pauses the cell cycle to allow time for DNA repair.
Checkpoint kinases
ATR, ATM
Regulate Chk1, Chk2
Can lead to apoptosis via p53 (Chk2, ATM), rev cell cycle arrest (Chk1,2), DNA repair enzyme prod (Chk1)
Thymine dimer repair
photolyase
NER
Uracil repair (from deamination of cytosine)
BER
Bulky adduct repair
NER
