Molecular mechanisms of DNA damage Flashcards
The SF 2 (surviving fraction at 2 Gy) for an irradiated population of cells is most closely correlated with the:
A. Level of gamma-H2AX 30 minutes after irradiation
B. Level of gamma-H2AX present 24 hours after irradiation
C. Acetylation of H2AX on lysine 4
D. Rate of DNA single-strand break repair
E. Rate of thymine glycol repair
B
The nucleosome contains an octamer of core histones: H3, H4, H2A, and H2B. Histone variants and their post-translational modifications regulate chromosomal functions; the post-translational modifications include acetylation, methylation, and phosphorylation. Histone H2A has nine subtypes, among them the H2AX variant, which is involved in the response to DNA damage. Production of DNA double-strand breaks (DSBs) by ionizing radiation leads to the rapid phosphorylation of histone H2AX on serine 139 (gamma-H2AX). The specificity of this reaction provides a reliable yardstick for DSBs and the means to spatially localize DSBs within the nuclei of cells (the gamma-H2AX focus assay). The degree of H2AX phosphorylation measured at a specific time after induction of the DSBs represents a balance between the rate of phosphorylation following DNA damage and the dephosphorylation that occurs as DNA repair progresses. SF2, the cell surviving fraction after 2 Gy, is a model-independent measure of radiation sensitivity. The numbers of phosphorylated gamma-H2AX foci shortly after the irradiation represent the initial level of DNA damage, but the number of phosphorylated gamma-H2AX foci at 24 hours after irradiation represent the residual level of unrepaired DNA double strand break at this time. It has been shown that the number of phosphorylated sites remaining 24 hours after irradiation directly correlates with intrinsic radiosensitivity. In contrast, after a 30 minute incubation, H2AX has been phosphorylated, but there has been little time for repair. A correlation between cell survival and the repair of either DNA single-strand breaks or thymine glycols has not been observed.
Which of the following statements about ionizing radiation (IR) induced DNA damage is correct?
A. IR causes only DNA double-strand breaks
B. IR may produce thymine glycols, but much less frequently than DNA double strand breaks
C. IR can cause more clustered lesions at low dose rates than at high dose rates
D. IR cannot cause oxidization of nucleotide bases
E. IR is unlikely to produce pyrimidine dimers
E
In contrast with the other forms of damage listed, pyrimidine dimers are principally produced following absorption of photons in the ultraviolet (UV) wavelength range and are not produced by X-rays. Pyrimidine dimers are cytotoxic, but more of these DNA lesions are required in order to achieve cell death compared to the DNA lesions produced by X-rays. It is estimated that the number of DNA lesions per cell from X-rays necessary to kill 63% of the cell population (thereby allowing 37% to survive) is 40 double-stranded DNA breaks (DSBs). In comparison, 1,000,000 pyrimidine dimers from ultraviolet radiation are needed to kill 63% of the cell population. IR can produce not only DSBs, but also other forms of damage including single strand breaks, thymine glycols, and base damage. These other forms of DNA damage, however, are more readily repaired and are less likely to result in cell death.
A clustered lesion:
A. Results from the creation of multiple DNA double-strand breaks (DSBs) within a particular exon of a gene following exposure to high LET radiation
B. Involves the formation of several DNA lesions within a highly localized region of DNA
C. Occurs more frequently as the LET of the radiation decreases
D. Represents the repair of multiple lesions within a gene
E. Results from transcription-coupled DNA repair
B
A clustered lesion, which has been hypothesized to play an important role in cell lethality, involves the formation of several DNA damages within a highly localized region of DNA.
Which one of the following assays would be the most appropriate to use for quantitative measurement of DNA double-strand breaks (DSBs) in cells immediately following exposure to ionizing radiation?
A. Alkaline elution
B. Western blotting
C. Neutral comet assay
D. PCR
E. BrdU incorporation assay
C
The neutral comet assay is used to measure DNA double-strand breaks (DSBs). The comet assay is the electrophoresis of single-cells in order to detect DNA damage and its repair. Cells are exposed to ionizing radiation, embedded in agarose, and then subjected to an electrical gradient to move the DNA into the gel. The negatively charged DNA in the cell moves through the agarose toward the positive electric pole. If there are no breaks, the cell’s DNA moves all together in a small ball. Double-strand DNA breaks create DNA fragments that are smaller than the unbroken DNA and migrate further into the agarose making what appears like a comet’s tail. Alkaline conditions cause the separation of the two strands of the DNA helix and allows the visualization of DNA fragments created by both double-strand and single-strand DNA breaks. In neutral pH conditions, the DNA helix is intact so single-strand breaks do not result in separate fragments and you can only see the fragments created by double-strand DNA breaks.
Which statement regarding radiation-induced nuclear foci is correct?
A. ATR is the main apical kinase that responds to radiation-induced double-strand breaks
B. ERCC1-containing foci indicate the presence of radiation-induced single-strand breaks
C. Gamma-H2AX foci can be detected within 15 minutes of radiation exposure
D. p53 forms ATR-dependent foci within minutes of radiation exposure
E. The remaining gamma -H2AX foci within the nucleus at 24 hours after irradiation is not related to the cellular sensitivity radiation.
C
One important characteristic of the cellular response to DNA lesions is the spatiotemporal manner by which repair and other proteins are recruited to the site of damaged DNA. Frequently, these protein accumulations can be visualized as subnuclear “foci” using immunofluorescence microscopy. Ionizing radiation-induced DNA double-strand breaks activate ATM kinase, which phosphorylates multiple damage response and repair proteins. ERCC1 is involved in nucleotide excision repair, in addition to roles in homologous recombination and replication fork repair but does not form subnuclear foci. Histone H2AX is phosphorylated by ATM within 15 minutes after irradiation and can be visualized using a phospho-specific antibody. These gamma-H2AX foci are regarded a marker for radiation-induced DNA double-strand breaks in cells. p53 itself does not form foci, though specific ATM-dependent phospho-forms of p53 might be detected as foci. ATM functions in response to double strand breaks. By contrast, ATR is activated during every S-phase to regulate the firing of replication origins, the repair of damaged replication forks and to prevent the premature onset of mitosis. Although ATR is activated in response to many different types of DNA damage including double strand breaks (DSB), a single DNA structure that contains a single-stranded DNA may be responsible for its activation. Furthermore, p53 does not form ATR-dependent foci. An elevated level of remaining H2AX foci at an extended time (such as 24 hours) after the initial DNA damage is indicative of an impaired DNA repair and cell sensitivity to radiation.
Which of the following has been shown to be a reliable surrogate marker for DNA double strand breaks (DSBs) in the cells?
A. Phosphorylated histone variant H2AX (or gammaH2AX)
B. Degraded histone H2AX
C. Dephosphorylated H2AX
D. Cleavage of Caspase 3
E. DNA methylation
A
The level of phosphorylated H2AX has been shown to correlate with the level of DNA double strand breaks.
