DNA Repair Flashcards
What is DNA Damage?
DNA is damaged thousands of times per cell per day
- due to many factors
- including basic chemistry of the nucleic acids
- external things like UV light, chemicals, radiation, pH, smoking, etc.
Allowing such cells to replicate is “bad”
State the short term consequences of DNA damage?
- Reduced proliferation
- Altered gene expression
- Cell death (apoptosis)
What are the long term consequences of DNA damage?
- aging
- diseases especially cancer
Is p53 a caretaker or gatekeeper?
p53 is the cellular gatekeeper for growth and division.
What is the importance of DNA repair?
DNA damage is common
Normal metabolism causes about 10^4 DNA adducts/cell/day through generation of endogenous oxidants (e.g. hydrogen peroxide, suoeroxxude etc.)
It can be recognized and efficiently repaired BUT,
Trying to replicate through it can convert DNA damage into mutations
Important to distinguish between DNA damage and DNA mutation
What is the molecular basis of mutation?
Mutations can be spontaneous or induced
Induced mutations are in addition to the rate of spontaneous mutation
There are two classes 9f spontaneous mutation- errrors of replication & spontaneous lesions
Differentiate the two types of spontaneous mutations?
- Errors of replication= mistakes made during replication
- only occurs during S phase of cell division - Spontaneous lesions- chemical changes that occur spontaneously
- Occurs in resting cell
Explain errors of replication. What is tautomerism?
Errors of replication- wring base is incorporated by DNA polymerase
-due to chemistry of the nucleotides
Tautomerism= the ability of certain chemicals to exist as a mixture of two interconvertable isomers
Thymine usually pairs A, the rare enolase form now pairs with G
Explain proofreading
Replication is very accurate but errors occur
DNA polymerase error rate= 1/100,000 bases
Actual error rate = 1 in 10,000,000
DNA Pol. enzyme has 5’ to 3’ polymerase activity AND 3’ to 5’ exonuclease activity
Also there are other repair systems that can detect and repair errors
What causes bloom syndrome?
Defect in a gene encoding a DNA helicase enzyme
-Required for replication repair, recombination
What are the characterist8cs if Bloom syndrome?
- smaller than average
- marrow chin, prominent nose and ears
- Fac8al rash (pigment and dilated blood vessels) upon exposure to sun (sometimes called a ‘butterfly’ rash
- often get diabetes and have neurological, lung and immune system deficiencies
- Chromosomal instability resulting in many chromosomal breaks and sister chromatid exchanges
- Higher risk of a broad range of cancer types
Increased sister chromatid exchange in bloom syndromes
Explain spontaneous lesions
Changes that occur in a resting cell due to the chemical nature of the DNA
Extremely common= tens of thousands of mutation events per cell per day
Exposure to mutagens such as sunlight, radioactivity, ionizing, radiation, specific chemicals may increase the rate of this type of DNA damage
Three main types of spontaneous DNA damage:
- depurination
- deamination
- oxidative damage
Explain depurination as a form of spontaneous lesions
Most common form of spontaneous lesions (about 10,000 each day/cell (about 1 in every 10 seconds)
Breaking of glycosidic bond between base and sugar in purine nucleotides
Sugar-phosphate backbone remains but base is lost
If it persists through replication then mutation can occur
Explain deamination as a type of spontaneous lesions
Very common about 5000 a day
Loss of amine group from a base (we can use cytosine as an example)
-cytosine (which base pairs with G) deaminates to form uracil (Uracul would pair with A)
This one is easy to fix- uracul doesn’t belong in dna
Why is 5-methyl cytosine a mutational hotspot?
5-methyl cytosine deaminates to form thymine, so this creates a T-G base pair
-Both of these bases are normally found in DNA; could be repaired back to a “C” (correct pair) or create a mutation “T”
So, repair machinery must guess
Why are methyl cytosine found in mutational hotspots?
Found in CpG islands as discussed in epigenetics
Not all positions are equally mutable, hotspots include things like 5-methyl cytosine or repeated baddd (AAAAA)
The point is that you can see the same mutation occurring at a relatively high frequency in specific pl@ces in the genome
Explain oxidative damage as a type of spontaneous lesion
A result of reactive oxidative compounds due to oxidative metabolism
-superoxides, peroxide’s etc.
Causes oxidative damage to many parts of cell including addition of oxygen groups to nucleotide bases
An example would be 8-oxo-7-hydroxyguanosine
Thus is oxidized to G will mispair with A and cause a potential TRANSVERSE MUTATION
What are mutagens?
These increase frequency 9f normal functions
What are common mutagens in our environment?
Serious
- uv (from sun. Or tanning beds)
- ionizing radiation
- aflatoxin (from moldy grain)
- benzene
- formaldehyde
- mustard gas
- anything that reacts with dna
Not serious:
Alcohol(aldehyde, break down product)
-bbq hamburgers(benzoapyrene)
Explain ionizing radiation
Include cosmic radiation, y-rays,x-rays, and radioactive particles (a,B,Y)
Relevant. To clinician and research scientists
But also relevant to everyone as we encounter a surprisingly large amount of radiation
- high energy particles or rays can cause many types of cellular damage up to and including death
- also causes extensive damage to DNA (base damaging type) including heritable mutations
Explain uv light as a mutagen
Generates several deleterious photo products
Such as cyclobutane pyrimidine dimers or 6-4 photo products
- covalent linkages. Between adjacent pyrimidine bases on the same strand
- these dimers will interfere with normal base pairing and block replication
These are known as pyrimidine dimers, or thymine dimers
What are the common repair mechanisms?
Most are indirect
Main mechanisms-but not intended to be comprehensive
Nucleotide excision
- removes more than a few (up to 30) base around a damaged site
- This is how pyrimidine dimers formed by UV damage are repaired
Base excision
-repairs a single (or just a few) damaged bases by removing it (damage by methylation, oxidation etc)
Mismatch repair(post replication repair)
- repairs mismatched bases
- that formed due to tautorism
Explain the excision repair mechanism
Mechanism is essentially the same fir all excision repair, just the size of the excised region varies
Involves:
Recognition of damage
Removal of damaged base or region around damaged base
The above 2 require specific. Repair proteins
Replacement
How does excision repair occur?
- Recognition of damage
- Recruit endonucleases
- Region excised
- DNA polymerase fills in gap
- Ligase seal nick
Explain the cause of Xeroderma pigmentosum
Autosomal recessive, biallelic pathogenic mutations in 9 different NER genes can lead to XP
So 9 complementation groups
Locus heterogeneity because there are many genes involved in the NER pathway
- XPA, ERCC3(XPB), XPC,ERCC2(XPD), DDB2(XPE), ERCC4(XPF), ERCC5(XPG), ERCC1, and POLH(XP-V)
- Recognize that XP is the common gene name for NER pathway components; other names don’t need to be memorized)
What is the incidence of XP?
Incidence:
- Europe-USA: 1:250,000
-Japan: 1:40,000=extreme sun sensitivity
What are the clinical features of Xeroderma pigmentosum ?
Clinical sun sensitivity
=sunburn, blistering, freckled with hyper pigmented skin lesions
Ocular involvement
-conjunctivitis, ocular tumors
Over 1000-fold increase in skin cancer including melanomas
- 20% have progressive neurologic degernation
- DNA Damage is cumulative and of course, irreversible
What is base excision repair?
Base nucleotides can be removed by DNA glycosylases which recognize specific damaged bases in DNA
Uracil glycosolase as a specific example for removal of U from DNA
Aberrant dna base removed by glycosylated, then sugar phosphate removed by endonuclease then base is replaced and ligated
There are different types of DNA glycosylases which are specific for different aberrant bases
What is mismatch repair?
Post replication repair mechanism
A form of excision repair (same basic mechanism, just different proteins involved)
Mismatched bases are recognized and excised
Also very important in relation to removing small repeats that tend to expand (e.g, triplet expansion disorders)
What are the steps in mismatch repair (a type of excision repair)?
Shows strand discrimination
- Mismatch missed by proofreading is recognized by MSH proteins
- Repair may occur during S-phase (if missed by proof-reading) or in G2 when genome is scanned for errors
- Excision of bases around mismatch
- Repair by re-synthesis
Many genes function in this pathway such as MSH2/6, MLH1-3, PMS2 + Several more
(It is enough that the MSH genes are the founding members =miss-match)
How does the cell know which is. The right strand in mismatch repair?
In prokaryotes it has been shown that hemimethylated strands are recognized. The methylatedstrand is the original and the unmethylated strand is the new one
In eukaryotes it may be more complex (of course). And interaction with replication machinery occurs but it also involves discernment of methylated/unmethylated DNA strands
What are frameshift mutations?
Tend to occur where there base repeats
- thought to result from slipping of DNA polymerase during replication of these repeats
- DNA loops or kinks at these points and one or more bases aren’t copied or are copied twice
Explain hereditary nonpolyposis for colon cancer
Autosomal dominant
- a result of mutations in genes encoding mis,at h repair proteins MSH2*, PMS1, PMS2, or MSH
- results in Microsatellites instability
Microsatellite instability frequently seen with these tumors= simple repetitive DNA tandem repeat sequences show size variability due to inaccurate replication
SSR
What are double stranded breaks?
A difficult type of mutation repair DSBs are particularly dangerous to dividing cells; high probability of loss of genet8c material of chromosomal rearrangements
What two mechanisms are used to f8x FNA double stranded breaks?
- Non-homologous end joining
- the more common method used
- doesn’t use homologous chromosome to repair the break - Recombination all repair
- doesn’t use homologous chromosome
- less error prone than NHEJ
Explain the effect of BRCA1 & BRCA2
Autosomal dominant breast cancer predisposition syndrome
Found in cells of breast and other tissue
-involved in DNA repair or apoptosis when DNA can’t be repaired
Women with BRCA1 or BRCA2 mutations have 85% risk 9f developing breast cancer by age 70
- And the risk of developing ovarian cancer is about 55% for BRCA1 and 25% for BRCA2 mutations
- this cancer risk increases as the person ages
- these are two different genes, so locus heterogeneity
Many hundreds of pathogenic variants have been identified in the BRCA1 gene, most associated with an increased risk of cancer (allelic heterogeneity)
Contrast somatic and germline errors
Somatic errors- can result in cancer
- does result in aging
- bad for you
Germline- can result in genetic disease visited on your descendants - bad for kids
It is thought that in gametes, one mutation every 10^8 nucleotides replicated, or about 60 mutations occur in every cell generation.
What are the consequences to mutations in DNA repair?
- Increased error rate
- Particularly true for proofreading and mismatch repair (MMR), producing “mutator” phenotype
-Damaged bases aren’t repaired efficiently
- Genomic instability
- mutations in gene involved in resting DNA repair and chromosome break repair will de-stabilize the genome e.g. Bloom syndrome, XP, many others
What is ataxia telangiectasia?
Defect is in ATM gene (11q22-23)
= a serine-threonine protein kinase with a number of functions including:
- detecting DNA damage (I.e. it is a sensor) and activating cell cycle arrest and DNA repair proteins(e.g. p53)
- autosomal recessive
- rare disease(incidence estimated to be 1:50,000)
- increased cancer risk (should avoid x-rays) affects cerebellum(=ataxia) and immune system
- ocular telaangiectasia common
DNA repair components…
All interact with each other
