Mechanisms of DNA Repair L1 AND L2 Flashcards
1
Q
Why have living things evolved DNA repair mechanisms? == 3
A
- Repair DNA ‘lesions’ (mutations), important to
REDUCE THE MUTATION RATE - May provide a SYSTEM TO BYPASS LESIONS so that REPLICATION AND CELL DIVISION can continue can create MUTATIONS (leading to natural variation)
- Most important REPAIR occurs while DNA is being
COPIED/SYNTHESISED…
2
Q
COMMON CAUSES OF DNA DAMAGE:
ENDOGENOUS VS EXOGENOUS
A
ENDOGENOUS:
(cellular metabolic processes)
1. mismatch of DNA bases
2. Hydrolysis
3. oxidation
4. alkylation
EXOGENOUS (environmental factors)
1. Ultraviolet radiation
2. ionising radiation
3. chemical agents
3
Q
COMMON CAUSES OF DNA DAMAGE: 4
A
- Replication stress = BASE MISMATCH
- oxygen radicals
ionising radiation
chemotherapeutics
= SINGLE-STRAND BREAK - ionising radiation, chemotherapeutics
= DOUBLE-STRAND BREAK, INTERSTAND CROSSLINKS - UV light, Polycyclic aromatic hydrocarbons
= BULKY ADDUCTS/ INTRASTRAND CROSSLINKS
4
Q
DNA Polymerases in DNA repair: 2
A
1 * Recall from DNA replication that there nare several types of DNA polymerases involved (sometimes different in
prokaryotes and eukaryotes)
2 * Many other DNA polymerases are also involved in DNA repair
5
Q
DNA polymerase enzymes in eukaryotes
A
– lots of them are there just for DNA repair
6
Q
General Statements on Repair Mechanisms = 3
generally, redundancy
A
- There are several complex pathways for DNA repair
- Generally:
MOST repair requires 2 DNA strands so that one can act as the template for synthesis of the other (to specify the base sequence)! - REDUNDANCY, ie most DNA damage can be repaired by more than one of the repair pathways.
- This ensures a low level of mutation, as if one
pathway fails to recognise/repair damage, another pathway may still act.
7
Q
Three basic principles of repair of DNA:
A
- direct reversal
- Base excision and replacement
- Segment removal and replacement
8
Q
SIX main mechanisms of DNA repair:
- Hydrolysis, Oxidation, Alkylation
A
- Bulky lesions OR base Modifications = DIRECT REVERSAL
- Single-strand break, Single-base damage = BASE EXCISION REPAIR (BER)
- Bulky lesions Crosslinks = NUCLEOTIDE EXCISION REPAIR (NER)
- Base mismatch = MISMATCH MEDIATED REPAIR (NMR)
- double strand break =
1– HOMOLOGOUS RECOMBINATION (HR)
2— NON-HOMOLOGOUS END-JOINING (NHEJ)
9
Q
Repair Mechanisms
—There are only two DNA repair mechanisms that DO NOT
require sequence homology from the complementary strand:
A
There are only two DNA repair mechanisms that DO NOT require sequence homology from the complementary strand:
- Direct reversal of damaged base
- Non-homologous end joining of double
strand breaks
10
Q
Direct Reversal of Damaged base….
A
Some lesions can be repaired by direct reversal, mediated by specific enzymes…
Example enzymes:
1. Photolyases
2. Alkyl transferases
11
Q
Direct Reversal of Damaged base —
Example enzymes:
- Photolyases:
A
Photolyases: only active in light, in dark need other
mechanisms repair UV induced damage to pyrimidines
-eg. pyrimidine dimer
12
Q
Direct Reversal of Damaged base —
Example enzymes:
- Alkyl transferases:
A
- Alkyl transferases:
remove unwanted alkyl groups on nucleotides
- transfer alkyl group to enzyme
13
Q
Direct Reversal of Damaged base - Example 1. Bacterial Photolyase
A
….repair requires light = photoreactivation
Uses energy ncaptured from light to break the covalent bonds in pyrimidine dimer
Thymine, Thymine …DNA backbone
—> UV light
Photodimer
—> photolyase + white light
Thymine, Thymine …DNA backbone
14
Q
Direct Reversal of Damaged base - Example
- Alkyl transferases = 4
A
1 * Remove alkyl groups on bases & transfer to
enzyme.
2 * System can be saturated by availability of
enzyme molecules (alkyl group accepted at
active site and enzyme becomes inactivated)
3 * Eg O6-methylguanine-DNA methyltransferase
- O^6=Methylguanine — methyltransferase —> Guanine + methyl (CH3)
15
Q
Repair Mechanisms
—All other DNA repair mechanisms DO require sequence homology from the complementary strand: —4—
A
All other DNA repair mechanisms DO require sequence homology from the complementary strand:
- Base excision repair
- Nucleotide excision repair
- Mismatch repair
- Homologous recombination to fix double strand breaks
16
Q
Base-Excision Repair…. WHY?
A
- Involves removal of a base & then the entire nucleotide is replaced
WHY?
Base is damaged, for example by spontaneous loss of the base, oxidation, or hydroxylation etc eg Base modifications
17
Q
Base-Excision Repair….STEPS = 5
A
- Each DNA glycosylase recognises and removes a specific type of damaged base, producing an apurinic or an apyrimidinic site (AP site)
- AP endonuclease cleaves the phosphodiester bond the 5’ side of the AP site…
- …and removes the deoxyribose sugar.
- DNA polymerase adds new nucleotides to the exposed 3’-OH group.
- The nick in the sugar-phosphate backbone is sealed by DNA LIGASE, restoring the original sequence.
18
Q
Base-Excision Repair….STEPS explanation…
A
- Each DNA glycosylase recognises and
removes a specific type of modified base by cleaving the bond that links the base to the 1’-carbon atom of a deoxyribose
sugar
- eg Uracil glycosylase removes
Uracil produced by Cytosine deamination - AP: apurinic or apyrimidinic sites (base lost)
3.AP Endonuclease cleaves the phosphodiester bond and other enzymes remove the deoxyribose sugar (dRPdeoxyribosephosphodiesterase removes
the deoxyribose phosphate group from
the AP site)
4.DNA Pol adds new nucleotides to the
exposed 3’-OH groups
- Nick in sugar-phosphate backbone
sealed by DNA ligase
19
Q
slide 20
A
- Eukaryotes use DNA polymerase β to replace
excised nts, which has no proofreading ability
and tends to make mistakes. On average, one mistake per 4000 nucleotides inserted. - About 20,000 to 40,000 base modifications
per day are repaired by base excision, so DNA
polymerase β may introduce as many as 10
errors per day into the human genome.
3.Some AP endonucleases have the ability to proofread. When DNA pol β inserts a nucleotide with the wrong base, DNA ligase cannot seal the nick in the sugar–phosphate
backbone because the 3′-OH and 5′- phosphate groups of adjacent nucleotides are not in the correct orientation.
4.AP endonuclease 1 detects the mispairing and uses its 3′ → 5′ exonuclease activity to excise the incorrectly paired base. DNA pol β then uses its polymerase activity to fill in the
missing nucleotide. In this way, the fidelity of
base-excision repair is maintained.
20
Q
Enzymes involved in Base-Excision Repair:
A
1.DNA glycosylases
- AP endonuclease
3.Deoxyribophosphodiesterase (dRpase)
4.DNA polymerase
5.DNA ligase
21
Q
DNA glycosylases
A
- cleave base-sugar bonds, removing base.
This leaves behind either an apurinic, or an apyrimidinic site
(AP site)
Recall uracil glycosylase (removes accidental U’s
inserted into DNA).
22
Q
AP endonuclease
A
AP endonuclease cuts the phosphodiester bond
23
Q
Deoxyribophosphodiesterase (dRpase)
A
then cleans up the
backbone by removing a stretch of neighbouring sugarphosphates,
so
DNA polymerase can fill the gap.
24
Q
DNA polymerase
A
DNA polymerase can fill the gap.
25
DNA ligase
DNA ligase then seals the new nucleotides in place
26
EXPLAIN Nucleotide-Excision Repair (NER) = 4
1. Complex process requiring many proteins…
2. Removes bulky lesions, such as PYRIMIDINE DIMER DISTORTIONS* of the double helix, as well as many other mutations causing distortions in DNA structure.
3. HIGHLY conserved pathway from bacteria to man –
VERY important to maintain DNA fidelity!
4. *Recall that humans don’t have a photolyase
enzyme to do this by direct reversal
27
Nucleotide Excision Repair = simple steps - 5
1. Bulky lesion
2. DNA opened to form bubble.
3. Damaged DNA excised
4. DNA polymerisation
5. DNA ligation
28
Nucleotide-Excision Repair (NER) in detail....
1. DAMAGE TO THE DNA DISTORTS THE CONFIGURATION OF THE MOLECULE.
- Lesion (distortions in DNA helix) / damaged bases detected by complex of enzymes that scan DNA looking for “problems” = DETECTION
2. AN ENZYME COMLEX RECOGNISES THE DISTORTION RESULTING FROM DAMAGE.
--Assembly of multi-protein complex at site, including enzymes to separate the two strands at the site of damage & Single Strand Binding (SSB) proteins to stabilise the single strands
= SEPARATION OF STRANDS
3. THE DNA IS SEPARATED AND SINGLE STRAND BINDING PROTEINS STABILISE THE SINGLE STRANDS.
--On affected strand, DNA around the lesion (±30 nts) is cut out by cleaving the sugar-phosphate backbone = EXCISION
4. AN ENZYME CLEAVES THE STRAND ON BOTH SIDES OF THE DAMAGE.
----Part of the affected strand is peeled away so the gap can be filled using
undamaged strand as template & DNA polymerase / DNA ligase = REPAIR
5. PART OF THE DAMAGED STRAND IS REMOVED...
6. ...AND THE GAP IS FILLED IN BY THE DNA POLYMERASE AND SEALED BY DNA LIGASE.
29
NER in E. coli vs eukaryotes
--------------ECOLI---------------
1. uvrABC excinuclease removes a 12-nucleotide fragment of DNA
2. DNA polymerase I synthesises new DNA
3. Ligase joins DNA segments
--------------EUKARYOTES--------
1. Repairosome and TFIIH subunit unwinds DNA
FORMATION OF A BUBBLE
2. Rad3, SsI2 - EXCISION OF DAMAGED STRAND
3. 3'incision, 5' incision, - DNA SYNTHESIS AND LIGATION
4. DNA
30
NER in E.coli
---- UvrA and B recognise
the damage
-----UvrC makes incisions
of either side of the
damaged segment of
DNA
----- UvrD helicase removes the nucleotide fragment
-----------------------------------
1. uvrABC excinuclease removes a 12-nucleotide fragment of DNA
2. DNA polymerase I synthesises new DNA
3. Ligase joins DNA segments
31
NER in eukaryotes:
1. Respirasome 20-30 proteins!
2. Transcription Factor IIH (10 subunit protein
complex). Helicase activity to unwind DNA around the damage region, verifies DNA damage
3. Rad3 is a protein kinase in yeast (human XPD protein homolog). DNA damage recognition and verification, also essential
for DNA unwinding at the damage site.
4. Ssl2 is related to the human XPB protein,
is a helicase, contributes to unwinding of DNA around damage site, works alongside
Rad3
5. 3’ incision by Rad2 endonuclease in yeast
(XPG in humans). 5’ incision by Rad1-
Rad10 endonuclease (XPF in humans)
------------------------------
1. Repairosome and TFIIH subunit unwinds DNA
FORMATION OF A BUBBLE
2. Rad3, SsI2 - EXCISION OF DAMAGED STRAND
3. 3'incision, 5' incision, - DNA SYNTHESIS AND LIGATION
4. DNA
32
Eukaryotic Nucleotide-Excision Repair (NER)
there are 2 Sub pathways for NER:
----Since both replication blockage and stalled
transcription can activate nucleotide-excision
repair
1.- Global genomic repair (GGR or GG-NER) –
Identify and repair DNA damage throughout the
entire genome
2. - Transcription-coupled repair (TCR or TC-NER)
– Selective for transcribed DNA strand in
expressed genes
---------They start differently, finish the same …
33
For eukaryotes: Coupling Excision Repair with Transcription...WHY?
1. Actively transcribed genes are repaired quickly, on the template strand. This is important so mRNA sequence is perfectly maintained
2. NER is coupled to transcription and is activated when RNA polymerase stalls at a lesion
3. WHY?
- Lots of eukaryotic cells don’t divide and so
DNA can’t be repaired in same way as bacteria,
i.e during replication. It makes sense to focus
repair effort only on those genes that are actually
being USEFUL to the cell, ie. Transcribed.
34
NER in eukaryotes: DAMAGE DETECTION, DAMAGE VERIFICATION, DAMAGE REMOVAL, REPLACEMENT
Genes – XP-A, B, C, D, E, F, G
Mutations in any of these seven genes can give rise to Xeroderma pigmentosum
XPC- Xeroderma pigmentosum C
XPC – Early sensors of
DNA damage within the
GG-NER pathway
XPB & XPD = helicases,
parts of TFIIH (10 subunits)
RPA = SS DNA-binding
protein
XPF = 5’-incision
XPG = 3’-incision
35
EXPLAIN: Eukaryotic Nucleotide- Excision Repair (NER) 2 pathways
1. GGR – either strand of DNA is damaged
GLOBAL DENOMIC REPAIR (GGR)
----Recognition of damaged base (XPC/R23B)
---- TFIIH
ENDS WITH CS = Cockayne Syndrome
XP = Xeroderma Pigmentosum
2.TC-NER, transcribed
strand damaged
transcription-coupled NER
---- RNA POLYMERASE
----RECOGNITION OF STALLED TRANSCRIPTION COMPLEX
---CSA, CSB
----TFIIH
---RN Polymerase, CSA, and CSB
ENDS WITH :
CS = Cockayne Syndrome
XP = Xeroderma Pigmentosum
36
Genetic diseases of NER:
Xeroderma pigmentosum (XP) = 4
1. autosomal recessive
condition
2 * Abnormal skin pigmentation and acute sensitivity to sunlight.
3.* Strong predisposition to skin cancer, incidence 1000- 2000 times that found in unaffected people.
4 * UV from sunlight, produces pyrimidine dimers in the DNA of skin cells. Defective NER cannot correct this
37
Genetic diseases of NER -
* Cockayne Syndrome (CS) = 10
1. Type 1 (CS-A) Life expectancy 10-20 years (25% of cases), present around 2-3 years
2 * Type 2 (CS-B) Life expectancy up to 7 years
(75% of cases), present at birth, brain development ceases
3 * Type 3 Late-onset or adult-onset, Life expectancy 40-50 years
4* Growth retardation
5 * Neurological abnormalities
6* Photosensitivity
7 * Eye Abnormalities eg cataracts
8 * Skeletal abnormalities eg joint contractures
9 * Premature aging
10 * No cure
38
Mismatch Repair...Mismatch repair systems have to: 3
1 * recognise mismatched bases by detecting
distortions in DNA
2 * determine which base is the correct one
3 * excise incorrect base & repair
39
MISMATCH REPAIR ....Sometimes the wrong base is added in DNA
replication…BUT - How to determine which is the correct base?
If an error in replication caused mismatch then the new strand
carries the error.
In bacteria DNA is methylated, but there is a
delay.
During this lag, mismatch repair can occur.
40
Mismatch Repair: Fixes Mispaired Bases: 3
1. Recognises mismatch
2. Removes the incorrect nucleotide on the new DNA strand (& nearby nucleotides)
3. Gap in new strand is filled in by DNA polymerase &
DNA ligase
41
Mismatch Repair in E. coli: New DNA Strand
Identified by Lack of Methylation
1. MutS- Mismatch recognition protein
ATP-dependent (MutS ATPase activity)
2 * MutL- facilitates assembly of functional
MMR complex, recruits and activates
MutH (ATPase acitivty)
3 * MutH- recognizes hemimethylated
dGATC sequence, incises unmethylated
strand, initiation site for excision
4.Adenine methylase - methylates A in GATC sequences
- this takes a few minutes!
42
Mismatch Repair in E. coli: New DNA Strand
Identified by Lack of Methylation STEPS = 5
1. in DNA replication, a mismatched base was added to the new strand
2. Methylation at GATC sequences allows old and newly synthesised nucleotide strands to be differentiated; a lag in methylation means that immediaetly after replication, the old strand will be methylated but the new strand will not.
3. The mismatched-repair complex brings the mismatched bases close to the methylated GATC sequence, and the new strand is identified.
4. Exonucleases removes nucleotides on the new strand between the GATC sequence and the mismatch.
5. DNA polymerase then replaces the nucleotides. correcting the mismatch, and DNA ligase seals the nick in the sugar-phosphate backbone.
43
Model for Mismatch Repair in Eukaryotes
- MutSa – recognizes
1-2 nt.
- MutSb - recognizes
larger mispairs.
-* hMutS- MutS homolog Mismatch
recognition protein
-How the old and
new strands are
recognized is not
known – Asymmetric loading of PCNA
(proliferating
cellular nuclear antigen) will indicate the nascent
strand?
- MutLa possesses a
PCNA/replication
factor c (RFC) dependent endonuclease activity, critical for 3’ nick-directed
MMR involving
EXO1
44
Double-strand breaks
What happens if both DNA strands are damaged so that one cannot act as a template in repair?? = 4
1. Ionising radiation results in double strand breaks
2. Interstrand crosslinks result when the two DNA
strands become covalently bonded – this is highly toxic as it causes replication blocks.
3. Some chemotherapeutic drugs cause crosslinks.
4. It is thought that crosslinks are repaired following double strand breaks on both sides of the crosslink
45
HOW Double-Strand Breaks = 3
1. Many of the same proteins are involved in homologous recombination & in DS break repair.
2. DS broken ends can be very dangerous - potentially harmful chromosomal rearrangements …
3. DS breaks can arise spontaneously
46
Repair of Double-Strand Breaks - Two distinct mechanisms to repair these:
1. nonhomologous end-joining (NHEJ)
2. homologous recombination (HR)
47
Explain Nonhomolgous End-Joining (NHEJ)
1 * Many repair mechanisms act in S-phase when DNA is
replicating, but many higher eukaryotic cells don’t replicate that often, so we need a way to repair DNA damage in G1 phase of cell cycle…
2 * So, if DS breaks occur in G1, there are no
complementary DNA strands to use, but need to repair, even if make mistakes. Better to rejoin free ends, even with errors, than to leave the DNA broken.
48
NHEJ involves : 4
1. Recognise damage
2. Binding of broken ends by KU70 & KU80
3. Trimming the ends to get 5’-P & 3’-OH
4. Ligation by DNA ligase IV
49
NON-HOMLOGOUS END-JOINING (NHEJ)
Don’t have the option to use the sister chromatid or homologous chromosome as a template
50
NON-HOMLOGOUS END-JOINING (NHEJ) STEPS
STEP 1--------------------------* 1. Ku Proteins –heterodimer Ku70 and Ku80
2 * Come in contact with ssDNA
3 * Slide on and back several base pairs
4 * Provides platform for other proteins to come and do their job
STEP 2 ---------------------------
1.* Ku proteins Recruit DNA-Protein Kinase catalytic subunit (DNA-PKcs)
2 * DNA-PKcs recruits Artemis protein and phosphorylates it to activate Artemis
51
Explain Homologous Recombination: 6
1. Utilises sister chromatid or homologous
chromosome to repair DS break.
2. Hence repair is
usually error-free.
3. Synthesis-dependent strand annealing (SDSA)
4.* Can use sister chromatid if not damaged
5 * Can use homologous chromosome, 95% sequence homology
6 * Happens when cells are dividing – S and G2
phases
52
Homologous Recombination STAGES INCLUDE: - 5
1 -Binding broken ends
2 -Trimming 5’ ends to expose SS regions
3 -Coating these regions with proteins forming
nucleoprotein filaments
4 -Search undamaged sister chromatid for
complementary sequence to use as template
4 -Joint molecule formation between
homologous damaged & undamaged duplexes
5 -Missing sequences then copied!
53
Homologous Recombination
STEPS 5 WITH SMALLER STAGES
------------STEP 1
1 * MRN Complex encounters damaged DNA
2 * Bind to 5’-ends and make a dissection of ~1000bp
3 * Left with 3’-overhangs
------------STEP 2
1 * ssDNA Nucleofilament
2 - RPA binds to the 3’-overhangs
3 - Protects overhangs from nucleases
4 - Prevents recoiling of the single strands
-----------STEP 3
1 * RAD51 with the help of BRCA2 replaces RPA
2 * RAD51 searches for homologous DNA
3 * RAD51 helps in the process of strand invasion
------------STEP 4
1 * RAD51 helps in the process of strand invasion
2 * D-Loop (Displacement Loop) formed
3 * DNA Pol uses red strand as a template and adds nucleotides tp the 3’-overhangs
4 * Stop signal is the intact DNA
--------------STEP 5
Cross over can be either good or bad depending on
context
54
Repair System:
MISMATCH
Type of Damage repaired?
REPLICATION ERRORS, INCLUDING MISPAIRED BASES AND STRAND SLIPPAGE.
55
Repair System:
DIRECT
Type of Damage repaired?
PYRIMIDINE DIMERS; OTHER SPECIFIC TYPES OF ALTERATIONS
56
Repair System:
BASE EXCISION
Type of Damage repaired?
ABNORMAL BASES, MODIFIED BASES, AND PYRIMIDINE DIMERS
57
Repair System:
NUCLEOTIDE EXCISION
Type of Damage repaired?
DNA DAMAGE THAT DISTORTS THE DOUBLE HELIX, INCLUDING ABNORMAL BASES, MODIFIED BASES, AND PYRIMIDINE DIMERS.
58
Repair System:
HOMOLOGOUS RECOMBINATION
Type of Damage repaired?
DOUBLE STRAND BREAKS
59
Repair System:
NON-HOMLOGOUS END JOINING
Type of Damage repaired?
DOUBLE STRAND BREAKS
