DNA repair mechanisms Flashcards
Can produce a covalent linkage between adjacent pyrimidine bases in DNA to form a thymine dimer
UV radiation
Repairs spontaneously-occurring DNA base modifications such as depurination and deamination, in addition to oxidation and alkylation base damage.
Base Excision Repair (BER)
Repairs bulky, helix distorting DNA lesions
Nucleotide Excision Repair (NER)
Repairs single-nucleotide mismatches and small insertion deletion mispairs
Mismatch repair (MMR)
The most frequent chemical reactions that can cause damage to DNA in cells are
Depurination and Deamination
Can release guanine as well as adenine from the DNA.
-This reaction occurs spontaneously when their N-glycosyl linkages to deoxyribose hydrolyze.
Depurination
How many purine bases are lost to depurination each day?
An estimated 5,000
Converts cytosine to uracil
Spontaneous deamination
In the care of depurination and deamination, the DNA backbone remains intact. The alterations in the bases are detected and repaired by the
Base excision repair pathway (BER)
In the BER, repair is initiated by enzymes, each of which recognizes a specific type of altered base in the DNA and catalyzes its hydrolytic removal. These enzymes are called
DNA glycolases
Once the damaged base is recognized, the DNA glycosylase reaction creates a deoxyribose sugar that
Lacks its base
This “missing tooth” is recognized by an enzyme that cuts the phosphodiester backbone and removes the damage. This enzyme is called
AP endonuclease
To replace the excised nucleotide, DNA polymerases execute repair synthesis. The final step of any DNA repair pathway is to seal the lingering DNA strand break or nick with
DNA ligase
Inherited mutations in the BER genes have not been
observed
Discontinuities in one strand of the DNA double helix and are usually accompanied by the loss of a single nucleotide and by a damaged 5’- and/or 3’-termini at the site of the break.
-considered to be a specialized sub-pathway of the BER
Single Strand Break Repair (SSBR) pathway
One of the most common source of SSBs is
Oxidative attack by Rective Oxygen Species (ROS)
Which is more common (by three orders of magnitude) SSBs or DSBs?
SSBs
Disintegration of oxidized deoxyribose is primarily detected by?
-Rapidly binds to, and is activated by, DNA strand breaks
PARP1
Serves as a molecular scaffold for multiple repair proteins, and stimulates multiple enzyme components of SSB repair
-accelerates the overall SSBR process
X-ray repair cross-complementing protein 1 (XRCC1)
Consequences of unrepaired SSBs include the following:
Collapse of dNA replication fork during S phase (forming DSBs); stalled transcription; increased cell death through PARP1 activation
Which polymerase fills the SSB?
Polymerase β
A rare autosomal recessive spinocerebellar ataxia syndrome that resembles ataxia telangiectasia, but lacks the non-neurologic features such as immune deficiency and telangiectasias.
Ataxia Oculomotor Apraxia (AOA1)
The characteristic features of AOA1 seem to be variable onset (1-16 years)
Cerebellar atrophy, ataxia, late axonal peripheral neuropathy, and oculomotor apraxia
The shaky and unsteady movements that result from the brains failure to regulate the body’s posture and the strength of direction movements
Ataxia
Ataxia is most often caused by disease activity in the
Cerebellum
A neurological condition characterized by loss of the ability to perform activities that a person is physically able and willing to do
Apraxia
Limited eye movement on command
Oculomotor apraxia
Has other features include cognitive impairment, hypercholesterolemia, hypoalbuminemia, and involuntary movements.
Ataxia Oculomotor Apraxia (AOA1)
The gene mutated in AOA1 was identified and designated as
Aprataxin (APTX)
Has putative repair mechanism for 5’ breaks (a DNA “End Processor”).
Aprataxin (APTX)
Unlike ataxia telangiectasia, AOA1 cells are only mildly sensitive – if at all- to
Ionizing radiation
There is no increase in cancer seen with this syndrome, nor increased chromosomal instability following ionizing radiation.
AOA1
Might be more dependent on APTX for DNA end processing than other post-mitotic cells, owing to a more limited availability of alternative end-processing factors.
-One reason why AOA1 might target nervous system
Neurons
AOA1 may also be largely restricted to the nervous system because of the
High levels of oxidative stress encountered by the nervous system
The limited regenerative capacity of neurons, compared with other non-cycling cell types that are more readily replaced by precursors, might render this tissue particularly sensitive to
Cell dysfunction or loss
Consists of a series of reactions that can repair damage caused by any large change in the structure of the DNA double helix.
NER pathway
In this pathway, a multienzyme complex scans the DNA for a distortion rather than for a specific base change or nick
Nucleotide Excision Repair (NER)
In the NER, once the bulky lesion has been found, the phosphodiester backbone of the abnormal strand is cleaved on both sides of the distortion, and an oligonucleotide containing the lesion is removed from the helix by a
DNA helicase
Broadly speaking, mutations that affect the repair process of non-transcriptionally active regions (the global genomic nucleotide excision repair pathway, GG-NER) are associated with
Skin Cancer
Mutations that affect the transcription-coupled nucleotide excision repair (TC-NER) pathway contribute to more
Developmental and Neurological disorders
The major damage recognition proteins for the GG-NER pathway
Xeroderma pigmentosum protein (XPC) or XPE
Mutations in XPC or XPE lead to
Non-neurological Xeroderma Pigmentosum
Non-neurological Xeroderma Pigmentosum is typified by
-these patients DO NOT have neurodegenerative symptoms
Extreme solar sensitivity and increased risk of skin cancer (2000x)
XPC and XPE detect
Helical distortions within the DNA
Mutations in XPC result in reduced capacity for the repair of lesions such as
Cyclobutane pyrimidine dimers
Most mutations in XP-C are inactivating null mutations, indicating that XP-C disease is largely due to a
Loss of enzyme function
Even low levels of XP-C protein produced by splice site mutations are enough to reduce UV sensitivity and create a milder phenotype. This suggests that levels of GG-NER gene products in normal cells are in
Excess of what is actually needed
A rare autosomal recessive genetic disorder characterized by numerous skin abnormalities ranging from excessive freckling to multiple skin cancers.
-The first DNA-repair disorder to be identified
Xeroderma Pigmentosum (XP)
Patients with XP with mutations in the GG-NER pathway develop symptoms at a median age of 1 to 2 years, although onset after 14 years old occurs in
5% of patients
Sixty to ninety percent of patients with XP develop
Ocular abnormalities
Do individuals that are heterozygous for XP have symptoms?
No
Patients with XP also have a 10- to 20-fold increase in the incidence of internal neoplasms such as
Brain, lung, and gastric tumors
Patients with mutations within the common pathway of NER such as XP-D and XP-A display both
Exterme sun sensitivity AND neurodegeneration
Caused by mutations in CSA or CSB, proteins that recognize DNA damage in transcriptionally active regions.
Cockayne Syndrome (CS)
The helix distortion caused by the DNA damage blocks RNA polymerase II progression, and the stalled RNA polymerase helps to initiate the
TC-NER repair process
The diagnosis of Cockayne Syndrome (CS) depends on the presence of what three signs?
- ) Growth retardation
- ) Abnormal sensitivity to light (photosensitivity)
- ) Prematurely aged appearance (progeria)
Patients with CS show severe developmental and neurological symptoms, but despite their photosensitivity, they do NOT manifest
Cancer
CS patients are often normal at birth but experience failure of brain growth and progressive neurologic dysfunction manifested by developmental delay, as well as behavioral and intellectual deterioration due to
Demylination of Neurons
One explanation for the differences in CS when compared with XP is that transcription does not recover after RNA polymerase is blocked. Furthermore, the arrested RNA polymerase is a potent inducer of
Apoptosis
A DNA repair pathway that is conserved from bacteria to humans
-Gretly improves the fidelity of replication (50-1000 fold)
MMR
A Hallmark of many MMR-deficient cells is instability at
Microsatellite regions (Microsatellite instability [MSI])
Repetitive DNA sequences of 1-4 base nucleotides that are particularly susceptible to DNA replication erros when the MMR system is absent
Microsatellites
In cancer cells, the presence of a defective MMR system leads to the accumulation of unrepaired mitotic errors, which occur more frequently in repetitive DNA tracts, mainly owing to the
- ) Slippage of error prone DNA polymerase
2. ) The occurrence of DNA helix hairpins at the replication fork
Widely used as a diagnostic marker for loss of MMR activity in tumor cells
MSI
The polymerase chain reaction (PCR)-based analysis of microsatellite regions is based on the comparison of their presence and length among patients or between
Normal and malignant cells
A microsatellite is considered unstable if the distribution of the fragments from the tumor sample differs from that of the
Normal tissue
Only 3-4% of colon cancers are
Hereditary
The most common inherited colon cancer characterized by an increased risk of colorectal cancer and other cancers. It is a common autosomal dominant syndrome characterized by early onset (average age
Lynch Syndrome (aka hereditary nonpolyposis colorectal cancer HNPCC)
The occurrence of neoplastic legions in tissues including endometrial, skin, ovarian, gastric, and renal, are an indicator of
Lynch Syndrome
The diagnosis of Lynch syndrome can be determined using the Amsterdam Criteria I, II and then by molecular genetic testing for germline mutations in
Mismatch repair genes
A hallmark of Lynch syndrome is
MSI
A particularly severe threat to genome stability that, if left unrepaired, could cause loss of chromosome fragments during mitosi, or chromosomal translocations that induce neoplastic transformation
Double-stranded breaks (DSBs)
To prevent loss of genetic material during mitosis cells
Arrest cell cycle at G2/M boundary until DSBs are repaired
If a tumor suppressor gene loses heterozygosity, the cell can eliminate the dangers of the DSBs by inducing
Apoptosis
Common DNA lesions induced by many types of stress and exposure, including ionizing radiation, oxidizing agents, replication errors and certain metabolic products.
DSBs
Which types of drugs can induce DSBs?
Certain antineoplastic drugs such as bleomycin, anthracyclines, and topoisomerase inhibitors
DSBs can be visualized by immunochemical staining for foci enriched in
Phosphorylated histone H2AX
DSBs can be repaired by which two fundementally different mechanisms?
- ) Non-homologous end joining
2. ) Homologous recombination
In mammalian cells, this repair pathway is probably restricted to the S and G2 phases of the cell cycle when a sister chromatid is present
Homologous recombination
Involves rejoining what remains of the two DNA ends, and the mechanism has evolved in a manner that tolerates nucleotide loss or addition at the rejoining site
Non homologous end joining (NJEJ)
An autosomal recessive, complex, multisystem disorder characterized by progressive neurologic impairment, cerebellar ataxia, variable immunodeficiency with susceptibility to sinus and lung infections, impaired organ maturation, x-ray hypersensitivity, ocular and cutaneous telangiectasia, and a predisposition to malignancy
Ataxia Telangiectasia (AT)
Small dilated blood vessels near the surface of the skin or mucous membranes. They can develop anywhere on the body but are commonly seen on the face around the nose, cheeks, and chin
Telangiectasias
The protein mutated in AT, that is normally activated by DNA DSBs and signals to the cell cycle checkpoint to slow the passage of cells through the cell cycle to facilitate DSB repair.
ATM
It seems likely that ATM also signals to the DNA repair machinery to facilitate
DNA repair
Subsequent studies suggest that, in the absence of ATM, persistent DNA DSBs are localized to
Heterochromatin
Seems to facilitate the entry of the DNA repair machinery into heterochromatin
-also responds to physiologic breaks in DNA during the development and differentiation of B and T cells
ATM
Up to 30% of AT patients develop
Lymphoid tumors
One of the most highly conserved groups of DNA helicases
-contribute to the maintenance of genome stability across various species
RecQ helicases
Defects in at least three of five human RecQ homologues are responsible for defined genetic diseases, and we refer to these as
Bloom’s syndrome (BS), Werner’s syndrome (WS), and RECQ4 syndromes
What are the RECQ4 syndromes?
Rothmund-Thompson syndrome (RTS), Rapadilino, and Baller-Gerol syndrome (BGS)
Interact with several proteins that have essential roles in DNA repair and may interact with specific DNA repair pathways
RecQ proteins
Has been implicated in BER of ROS induced damage, methylation-induced DNA damages and SSBs
WRN (A ReQ Helicase) (mutation of which causes Werner Syndrome)
Most of the mutations in WRN, a protein with ATPase, helicase, exonuclease, and single-strand annealing activities, lead to truncations of the protein, and/or elimination of a crucial nuclear localization signal that prevents WRN from finding its way to the
Nucleus
WRN has been shown to physically and functionally interact with key proteins involved in NHEJ and HR, which suggests an important role of WRN in
DSBR
A rare autosomal recessive disorder caused by mutations in the WRN gene
-Generally patients exhibit a stocky appearance
Werner’s syndrome
Generally, the first clinical sign of WS is a lack of
Pubertal growth during teen years
In one study, 95% of WS patients were reported to have
Short stature
In their 20’s and 30’s, patients with WS begin to manifest
Skin atrophy, loss of hair, and graying hair
Have telomere shortening, chromosomal rearrangements, increased susceptibility to malignant transformations and frequent telomere fusions.
WS cells
Mutations in which three genes give rise to the combined symptoms of the NER diseases XP and CS?
XPA, XPB, XPD, XPG, and XPF
The nervous system is very sensitive to
DNA damage
The neurological symptoms are almost the exclusive presentation of the disease in
DNA SSBR deficiencies
What may account for the sensitivity of the nervous system to DNA damage?
Brain metabolizes 20% of oxygen, but has a lower amount of antioxidants than any other part of the body
Neurons are particularly susceptible to
Reactive oxygen species (oxidative stress)
Active transcription can be estimated to involve only about 1–2% of total genomic DNA indicating that the lethal (apoptotic) signal generated by a failure of TCR of active genes must be about 50 to 100 times as potent per unit of repairable DNA, as is the signal for lethal events from a failure
GG NER
Might be a source for the pathological consequences in CS and cause cell loss from non-dividing tissues such as brain and retina
The potent apoptotic signal generated by failure of TC-NER
Apoptosis that removed damaged cells from the skin would conversely prevent UV-light carcinogenesis, especially in
CS cells
Oxidative damage has been reported in the brains of repair-deficient patients. Some tissues that degenerate in CS appear to be unusually sensitive to oxygen levels, including the
Purkinje and Retinal cells
The initial damage response in TC NER is mediated by the coupling factors
CSA and CSB
Has a repair mechanism for 5’ breaks
-mutation of this gene results in AOA1
Aprataxin (APTX)
Major damage recognition proteins for the GG-NER
XPC and XPE
WRN has been implicated in
Base excision repair of ROS oxidative damage, methylation induced DNA damage, SSBs, and DSBs
Ataxia Oculomotor Apraxia is due to a molecular defect in
Aprataxin (APTX)
What DNA repair pathway is affected by AOA1?
Single-stranded break repair pathway
Ataxia telangiectasia is due to a molecular defect in
AMT
What DNA repair pathway is affected by AT?
Double-stranded break repair
Cockayne syndrome is due to a molecular defect in
CSA or CSB
What do CSA and CSB do?
Recognize the stalled polymerase, remove it, and recruit other proteins to fix damage
What DNA repair pathways are affected by mutation in the ReQ Helicase WRN?
BER, SSBR, DSBR
Mutations in XPA and XPD are mutations that affect the common pathway. This disease is still referred to as
XP
