1
Q
How often is DNA being damaged and repaired?
A
- constantly being repaired in normal healthy cells to restore normal sequence
- up to 500,000 DNA modification events per day
2
Q
What kinds of things does the DNA face that can damage it?
A
endogenous- inside source (reactive oxygen, thermal changes, metabolism)
exogenous- outside source (chemicals, irradiation, UV light, x rays)
3
Q
When does a mutation result? vs healthy cell?
A
- when the rate of damage exceeds the rate of repair
- when rate of damage equals rate of repair = healthy
4
Q
Are all mutations bad?
A
no because some lead genetic variation which drives evolutionary change
5
Q
Mutations in germ cells cause what? somatic cells?
A
- germ- heritable genetic disorders
- somatic- acquired diseases such as cancer or neurodegenerative disorders
6
Q
T/F
Polymerase error is major source of DNA damage.
A
False
minor
7
Q
What are three types of DNA damage?
A
- depurination
- deamination
- oxidative damage to nucleotides
8
Q
What is depurination? How many times does this happen per day?
A
- spontaneous loss of adenine or guanine nitrogenous base
- 5,000 per cell per day
9
Q
What is deamination? How many times does this happen per day?
A
- common spontaneous loss of NH2
- results in unusual bases not usually found in DNA
- 100 per cell per day
10
Q
What happens in oxidative damage to nucleotides?
A
- over 80 types of base damage due to reactive oxygen species
- 8 hydroxyguanine (8-oxoG)
- base pairs with A instead of C
11
Q
What makes recognition and repair of deaminated bases easier?
A
the nucleotides formed are not normally present in DNA
12
Q
What does the deamination of methyl cytosine turn into? Why is this significant?
A
- thymine
- it is a normal nucleotide so it is difficult for the cell to recognize it as damage
- this is why CpG are underrepresented in the genome
13
Q
Deaminated adenine?
A
hypoxanthine
14
Q
Deaminated guanine?
A
xanthine
15
Q
Deaminated cytosine?
A
uracil
16
Q
Does thymine get deaminated? why?
A
no amine group
17
Q
What property of DNA makes it ideally suited for repair?
A
double stranded structure
18
Q
How many different proteins are involved in DNA repair?
A
over 100 different proteins
19
Q
What are the 5 major pathways for DNA repair?
A
- Mismatch Repair (MMR)
- Base Excision Repair (BER)
- Nucleotide Excision Repair (NER)
- Homologous Recombination (HR)
- Non-homologous end joining (NHEJ)
20
Q
Replication error is what type of damage (endo or exo)? What mutations can this lead to? What type of repair is used to fix it?
A
- endogenous
- base mismatches, insertion/deletion loops
- fixed by mismatch repair (MMR)
21
Q
Problems in the cell environment is what type of damage (endo or exo)? What mutations can this lead to? What type of repair is used to fix it?
A
- endogenous
- spontaneous deamination, depurination, oxidation
- fixed by Base excision repair (BER)
22
Q
UV exposure is what type of damage (endo or exo)? What mutations can this lead to? What type of repair is used to fix it?
A
- exogenous
- cyclobutane pyrimidine dimers, 6-4 photoproducts
- fixed by photo reactivation, nucleotide excision repair (NER)
23
Q
ionizing radiation is what type of damage (endo or exo)? What mutations can this lead to? What type of repair is used to fix it?
A
- exogenous
- single and double strand breaks, oxidation
- fixed by double strand break repair, BER
24
Q
Chemical exposure is what type of damage (endo or exo)? What mutations can this lead to? What type of repair is used to fix it?
A
- exogenous
- alkylation, interstrand crosslinks
- fixed by alkyl transferases, recombination repair
25
Mismatch repair (MMR) reduces error rate by how much? What type of damage does it repair? What is it important for?
- 100 times
- repairs polymerase error and small insertions nd deletions
- important for stability of micro satellites
26
Mutation of MMR enzymes is associated with what diseases?
- inherited colorectal cancer
- lynch syndrome
- HNPCC
27
What is the process of MMR? What is strand selection based on?
- proteins (MutS and MutL) scan for small structural distortions
- strand selection based on nicks in newly synthesized strand
- area between mismatch and nick is excised
- DNA pol fills gap
- DNA ligase seals nick
28
What is the biggest challenge for MMR?
making sure it repairs the correct strand, newly synthesized
29
What is the most common type of repair in human cells? what are the subtypes?
-excision repair:
- base excision repair (BER)
- nucleotide excision repair (NER)
- transcription coupled repair (TCR)
- global genome repair (GGR)
30
What are the basic steps in excision repair?
- recognize damage
- remove damage by excising part of the damaged strand
- fill in gap, using the other strand as template
- ligate to seal nicks
31
What are DNA glycosylases in BER? what types of damage do they recognize? What do they catalyze?
-family of enzymes that recognize specific types of damaged nitrogenous bases
- deaminated C's, A's
- alkylated or oxidized bases
- bases with opened rings
- bases with reduced double bonds
-catalyze the removal of the damaged nitrogenous base by hydrolysis of the N-glycosidic bond
32
What is the process of Base excision repair (BER)?
- damage to nitrogenous base portion of a nucleotide
- glycosylase removes base, backbone intact
- AP endonuclease cuts backbone 5' of AP (apurinic) site
- AP lyase (phosphodiesterase) removes sugar by cutting 3' of AP site
- DNA pol fills gap
- Ligase seals nicks
-AP sites from depurination fixed this way, but starts after glycosylase step
33
What type of damage does NER fix compared to BER?
- damage involves entire nucleotide, larger structural distortion
- covalent attachment of large hydrocarbons
- pyrimidine dimers- covalent bonds between pyrimidines, caused by UV damage
34
What is the process of NER?
- Excision nuclease (endonuclease) makes a double excision surrounding the damage
- DNA helices (TFIIH) removes the damaged section
- DNA pol fills gap
- DNA ligase seals nicks
35
What are the subtypes of NER?
- transcription coupled repair (TCR)
| - global genome repair (GGR)
36
What does TCR repair? What triggers it?
- removes lesions from transcribed regions of DNA
| - triggered by stalled RNA pol
37
What does GGR repair? It is decreased in what?
- removes lesions all over genome
| - decreased in terminally differentiated cells
38
When do double strand breaks occur? What can this type of damage lead to? What repairs double strand breaks?
- happen in every S phase
- can also be caused by ionizing radiation or oxidizing agents
- damage leads to chromosomal translocations or cell death if not repaired
- repaired by homologous recombination (HR) or non-homologous end joining (NHEJ)
- DNA fragments joined could be broken ends of same chromosome, or ends of 2 chromosomes
39
Non homologous end joining (NHEJ) is the dominant mechanism in what phase? why?
- G1 after mitosis
- there are no sister chromatids available in G
- quick and dirty mechanism
40
What is the process of NHEJ?
- ends bound by Ku, kinase, and nuclease (artemis)
- Po4- kinase unwinds the DNA
- ends anneal via short region with compatible sequence
- ends trimmed, extended as needed, then ligated
41
Why is NHEJ known as the error prone mechanism?
- several base pairs at the site of the break are frequently removed
- some DNA is deleted, but some DNA has already been lost from the double strand break
- repairing the break is more important than losing a few more nucleotides, especially if caused by ionizing radiation
42
The DNA fragments joined in NHEJ could come from what?
the same chromosome or the ends of 2 different chromosomes
43
Homologous recombination (HR) is the dominant mechanism in what phase? What is HR? Why is it essential? What else is it involved in? Precise mechanism?
- used for repair of double stranded breaks in S and G2 when sister chromatids are available, before cell has divided, mitosis
- HR is the genetic exchange between a pair of homologous DNA sequences, usually two copies of the same chromosome
- essential for every proliferating cell to restart stalled replication forks
- also involved in crossing over of chromosomes in meiosis- resolution of join molecule is different
- cleavage and rejoining are precise, no nucleotides lost or gained
44
How does the resolution of the joint molecule differ in mitosis vs meiosis?
mitosis repair pathway does not result in crossover, but meiosis does which increases genetic diversity
45
what type of junction forms in HR in meiosis?
holliday
46
What is the process of HR after a double strand break?
- nuclease digest 5' ends of broken strands
- strand exchange by complementary base pairing
- repair polymerase synthesizes DNA using undamaged DNA as template, strand invades
- invading strand released, broken double helix reformed
- DNA synthesis continues using strands from damaged DNA as template
- DNA ligation
47
What does HR require to happen?
- homolgous sequences
- strand invasion and exchange
- strand elongation
- cleavage and ligation
48
What are Brca 1 and Brca 2 involved in? Mutations of these are found where?
- involved in formation of joint molecules in HR, may be involved in NER and NHEJ
- mutated in inherited breast and ovarian cancer
49
How does a replication fork break? What repairs broken replication forks?
- if a replication fork reaches a single strand break, it collapses
- HR can be used to repair the fork using the newly synthesized DNA from the other strand
50
What is the process of repairing a broken replication fork?
- replication moves until it reaches a nick, then it breaks
- nuclease degrades 5' end of broken strand
- strand exchange, DNA synthesis
- strand breakage, more DNA synthesis
- replication fork restarts
51
What is xeroderma pigmentosum? What causes it? Risk of cancer?
- rare autosomal recessive disorder
- defective NER causes failure to repair UV damage
- 1000X increased risk of cancer in cutaneous basal and squamous cell carcinomas
- increased risk of internal cancer by age 20
52
What helped to identify proteins involved in pathway for xeroderma pigmentosum?
- locus heterogeneity
| - 7 complement groups (one group has proteins that another group doesn't have) compared proteins
53
What is the mutator phenotype?
mutations in proteins involved in cell growth eventually result in loss of cell cycle control
54
What are the caretaker proteins? why?
DNA repair proteins because they can be considered tumor suppressor proteins, repairing DNA before cancer takes over
55
what can faulty DNA repair mechanisms result in?
double strand breaks -> chromosomal rearrangements -> mutator phenotype -> mutation of tumor suppressor genes or oncogenes -> loss of cell cycle control checkpoints and progression -> genomic instability -> cancer
56
What is the process of chromosome breakage fusion bridge cycle?
1. cell enters S phase and replicates its DNA despite unprepared strand break
2. one daughter cell inherits a chromosome lacking a telomere
3. cell enters S phase and replicates DNA
4. sister chromatid ends that lack telomere fuse together
5. fused sister chromatids are pulled apart at mitosis, creating breakage at a new sit
6. one daughter cell inherits a chromosome with duplicated genes but again lacking a telomere
7. cycle repeats
57
How do cells normally treat a loss of telomeric DNA?
- similar to double strand breaks
- cell cycle checkpoints are activated, repair pathway stimulated
- if too much to repair, cell becomes senescent and dies
58
What is the consequence of the chromosome breakage fusion bridge cycle? why does this happen?
- checkpoints fail, cell continues to proliferate
- continues to lose telomeric DNA until telomeres are not functional
- chromosomes fuse together and undergo the cycle
- most of the time the cell dies due to genomic instability
- sometime rearrangements reactivate telomerase and chromosomes are stabilized
- cell survives but has many mutations
59
What can the reactivation of telomerase do?
restored ends of DNA and make the cell immortal, cancer
60
What are micro satellites? Where are they found?
- simple sequence repeats
- short, tandem repeats of 1-6 nucleotides
- most abundant class of repetitive DNA
- scattered throughout genome, more frequent in non coding regions
- presents challenges to replication and repair mechanisms
61
What are expansion of trinucleotide repeats known as?What do they cause? What does the mechanism of disease depend on?
- dynamic mutations
- at least 20 neurological or neuromuscular disorders are caused by them
- depends on location of repeats within the gene, and function of affected protein
62
What is the genetic anticipation of expansion of trinucleotide repeats?
- increased odds of inheriting
- decreased age on onset
- increased severity in later generations
63
What repeated sequence create stronger pairs?
CG sequences
64
What diseases result from trinucleotide expansions in non-coding sequences? large changes in repeat number are associated with what?
- Fragile X
- Freidrich ataxia
- spinocerebellar ataxias type 8 and 12
- myotonic dystrophy
-maternal transmission
65
What are two disease causing mechanism for trinucleotide expansion in non-coding sequences?
1. decreased transcription due to changes in chromatin structure or increased methylation (fragile X)
2. RNA gain of function- abnormal interaction of RNA with proteins involved in mRNA processing (myotonic dystrophy)
66
What diseases result from trinucleotide expansions in exonic repeats (polyglutamine diseases)? What are they due to? large changes in repeat number are associated with what?
- huntingtons disease
- spinobulbar muscular atrophy
- spinocerebellar ataxias types 1, 2, 3, 6, 7
- due to expansion of CAG- glutamine
- paternal transmission
67
What is the disease causing mechanism for trinucleotide expansion in exonic repeats?
toxic gain of function of mutant protein
68
What are the two models of trinucleotide expansion hypothesis?
1. slippage of newly synthesized strand during rapid replication
2. Recombination
- could be happening at same time
69
Hypothesis of slippage in expansion?
- primary hypothesis
- repeats tend to form hairpin structures in newly replicated lagging strand
- pulls polymerase back so it copies the same area twice
- should be repaired by MMR, but structural features of repetitive DNA make this more difficult
70
Hypothesis of recombination in expansion?
- repetitive sequences are recombinogenic
| - expansion can result due to unequal cross over and to polymerase slippage during repair of DNA synthesis
71
How many loci are unstable in an unstable micro satellite (MSI+)? what cancers result? what causes micro satellite instability and cancers? Can it be inherited? what else?
- at least 2 out of 5
- occurs in 15% of all colorectal cancers
- also in ovarian, endometrial, skin, brain, stomach cancers
-caused by defects in MMR system
- inherited in lynch syndrome, HNPCC
- somatic mutation
- methylation of gene encoding MMR proteins
72
Affected micro satellites (tumors) are usually what type of cells?
- diploid
- rarely show loss of genetic material or loss of heterozygosity
- improved prognosis
73
Do tumors respond the same to chemotherapy and/or radiotherapy?
- no
| - 5 fluorouracil selectively kills with MMR system intact, no survival benefit for patients with MSI-H tumors
74
How are MSI (micro satellites) and TNR (trinucleotide repeats) separate but related?
MSI- lynch syndrome/HNPCC, micro satellite changes throughout genome are not repaired due to mutations in MMR proteins
TNR- huntingtons, the secondary structure of DNA in a specific gene causes failure of MMR to repair expansion
75
Where are CpG islands normally found? Are they methylated? What are abnormal methylation patterns in cancer?
- found in promoters, not methylated
- most CpG in other areas of genome are methylated
- overally hypomethylation
- hypermethylation of CpG islands in promoters
76
Hypermethylation of CpG islands is associated with what?
- abnormal gene silencing
- recruitment of histone deacetylases (HDAC)
- inhibition of transcription
- inactivation of tumor suppressor genes or caretaker genes
77
global hypomethylation results in what? What does this cause?
- loss of imprinting (LOI)
| - causes imprinted genes to be over expressed by expressing both alleles so twice as much protein is made
78
Is methylation responsible for inherited cancer syndromes? Why?
no because methylation patterns are erased in the germ line cells
79
How can methylation be involved in sporadic cancers or the 2nd hit of inherited cancers? CIMP?
- methylation of a tumor suppressor could be second hit by inactivating normal allele in a somatic cell
- CIMP- CpG island methylator phenotype characterized by promoter hypermethylation (present in 18% of colorectal cancers)
MBM
flashcards
Decks in class (24)
# Cards
-
DNA Replication110
-
RNA and Protein Synthesis98
-
Regulation of Gene Expression133
-
DNA Repair79
-
Genetics 1165
-
Genetics 217
-
Proteins83
-
Hormones 161
-
Hormones 263
-
Energy Production and Utilization88
-
ATP Synthesis78
-
Carbohydrates 182
-
Carbohydrates 225
-
Glycogen42
-
Gluconeogenesis/Ketogenesis49
-
Lipids138
-
Fatty Acid Oxidation62
-
Fatty Acid Synthesis47
-
Lipoproteins65
-
Dietary Proteins 180
-
Dietary Proteins 246
-
Hemoglobin92
-
Hemostasis63
-
Nutrition38
