Mechanisms of DNA/chromosome damage Pt 2
Which of the following chromosomal aberrations is formed from a radiation-induced break in each arm of a single chromatid in G₁?
A ring chromosome aberration occurs when there are two breaks in the same arm of a pre-replication (G₁) chromosome(early in interphase prior to replication).
Which one of the following radiation-induced chromosome aberrations is a "single hit" type? Acentric ring, Anaphase bridge, Dicentric and terminal deletion
A terminal deletion is produced when a single chromosomal break results in deletion of a portion of the chromosome, that is, a "one-hit" aberration.
Which is attracted by MRN (Mre11-Rad50-Nbs1) during recognition of a DNA double strand break?
ATM, which initiates mutual phosphorylation with Chk2 and a phosphorylation cascade of many downstream targets to initiate DNA repair.
Chromosomal translocations produced by ionizing radiation are generated:
After the formation of at least 2 DNA double strand breaks The formation of a chromosome translocation between two chromosomes needs two DSBs. The dose response curve is linear quadratic because the two DSB can be produced by a single event or two independent events.
Which double-strand break DNA repair system utilizes PARP1 and Pol θ?
Alternative end joining (AEJ) plays a role in chromosomal aberrations and is more error prone than NHEJ. AEJ is used for larger lesions and gross abnormalities. Pol-theta and PARP1 are involved in AEJ. Pol-theta is a DNA polymerase → translesion DNA polymerase.
In NHEJ, which DNA repair protein with endonuclease activity is involved in processing strand breaks prior to DNA ligation?
Artemis
Ionizing radiation induced chromatid-type aberrations are a consequence of failed or mis-repaired DNA double strand breaks produced during which phase of the cell cycle?
Chromatid type of chromosome aberrations are caused by chromatid breaks in S or G2 phase. The breaks induced in G1 phase often display as chromosome type of aberrations.
53BP1 is stimulatory of which double-strand break DNA repair system?
Classical non-homologous end-joining 53BP1 is a p53 binding protein which also promotes NHEJ.
Which of the following cyclin proteins is synthesized in the M phase of the cell cycle?
Cyclin B is synthesized in G₂ and M. Cyclin A is synthesized in S and G₂, while cyclins D and E are synthesized in G₁.
Mismatch repair involves the action of a DNA glycosylase and an AP endonuclease, T/F?
False Base excision repair, not mismatch repair, involves the action of a DNA glycosylase and an AP endonuclease. People with Fanconi anemia are highly sensitive to DNA cross-linking agents due to inhibition of the mono-ubiquitination of FANCD2, a downstream Fanconi anemia protein, following genotoxic stress.
The BLM protein serves to coat single stranded DNA regions to prevent their degradation. T/F
False The BLM protein is a helicase. RecQ genes encode helicases that play a critical role in protecting replication forks. Decreased expression of RecQ genes results in aberrant DNA replication and genomic instability. RPA serves to coat single stranded DNA regions generated during homologous recombination to prevent their degradation.
UV usually induces purine dimers involving guanine bases. T/F
False UV causes the production of pyrimidine dimers in which a cyclobutyl ring is formed between adjacent cytosine and thymine bases
Several of the genes that are mutated in people with Fanconi anemia encode for products involved in which DNA repair system?
Homologous recombinational repair Patients with fanconi anemia have mutations in both copies of the fanconi anemia gene. D1 complementation group of fanconi anemia is the same gene as BRCA2. FA has difficulty maintaining replication forks. If truncation mutation (non-functioning), then person doesn't necessarily have FA but has increased risk for breast cancer. If hypomorphic mutation (less effective than protein in normal state, e.g. amino acid change) in both alleles, then can get FA.
What is the most common form of cell death from radiation?
In mitotic cell death (aka mitotic catastrophe), while attempting to divide, cells die due to damaged chromosomes. As a mode of radiation-induced cell death, apoptosis is highly cell-type dependent.
An accidental exposure to a radiation source is reported one month following irradiation of a person not wearing a dosimeter. Which of the following assays would represent the best method to estimate the radiation dose received by this person?
Karyotyping peripheral blood lymphocytes
The SF₂ (surviving fraction at 2 Gy) for an irradiated population of cells is most closely correlated with the
Level of γ-H2AX present 24 hours after irradiation 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. 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.
In which phase of the cell cycle are cells typically the most sensitive to radiation?
M phase Cells are usually the most sensitive during or close to mitosis. Cells are least sensitive at the latter part of the S phase and early G₂ phase. If cells have a long G₁ phase, they tend to be resistant in early G₁ and sensitive in late G₁. Cells during late G₂ are nearly as sensitive as cells in mitosis.
Which of the following is the best measure for the presence of radiation-induced chromosome aberrations in interphase cells?
Micronuclei which develop when a pseudo nuclear membrane forms around acentric chromosome fragments or whole chromosomes that did not segregate properly into daughter cells during the previous mitosis. Micronuclei are observed in peripheral lymphocytes and thus can be seen in interphase cells.
Hereditary non-polyposis colon cancer (HNPCC) is associated with a defect in:
Mismatch repair
The majority of reactive oxygen species in a cell are produced _____
Most reactive oxygen species in a cell are produced from mitochondrial respiration.
Which of the following phenotypic characteristics would be increased in cells that are defective in the FEN1 endonuclease, which is known to be involved in base excision repair (BER)?
Mutation rate UV, alkylating agents, and ionizing radiation generally do not produce the types of DNA damage repaired by base excision repair, such that a defect in the FEN1 endonuclease should not produce a particular sensitivity-associated phenotype. A BER defect will lead to an overall increase in genomic instability and mutation rate, however.
A PALB2 mutation is known to increase the risk of all of the following cancers
Partner and localizer of BRCA2, also known as PALB2 or FANCN, is a protein which in humans is encoded by the PALB2 gene. This gene encodes a protein that functions in double-strand break repair. PALB2 increases the risk of breast, pancreatic and ovarian cancer but not prostate cancer
What ATM-mediated epigenetic modification of histones occurs immediately after the production of radiation-mediated double strand breaks in DNA?
Phosphorylation Epigenetic regulation of histones can occur through acetylation, phosphorylation, methylation and ubiquitination. Phosphorylation of H2AX - gammaH2AX is a molecular marker of radiation-induced DNA double strand breaks that occurs with minutes after irradiation; and prior to strand annealing.
What accounts for the higher surviving fraction when delaying the subculture of quiescent cells in vitro after a large, single dose of X-rays compared to cells cultured immediately after irradiation?
Potentially lethal damage recovery
Which of the following catalyzes strand exchange during homologous recombination repair of DNA double strand breaks?
RAD51 is a human homologue of the RecA recombinase found in E. coli and mediates the invasion of the homologous strand of the sister chromatid. RAD52 provides protection against exonucleolytic degradation, while BRCA1 assists in the loading of RAD51. Artemis removes 3' and 5' overhangs during non-homologous end joining.
Which chromosome aberration is considered the hallmark of radiation exposure?
Reciprocal translocation Reciprocal translocation can be stable lesions (unlike micronucleus, terminal deletions and dicentric exchanges that are lethal or lost at division) and are therefore evident in cells many divisions after irradiation; consequently, they are considered a hallmark of radiation exposure.
Which of the following chromosome aberrations is most appropriate to use for detection of exposure to ionizing radiation following a possible irradiation?
Reciprocal translocations are relatively stable chromosome aberrations, and therefore can be detected at long times after exposure to ionizing radiation.
Which of the following is true regarding histones and radiosensitivity?
Removal of histones increases radiosensitivity Chromatin, histones and proteins protect DNA from damage, so the removal of histones enhances sensitivity to radiation.
Which type of chromosome aberration predominates following exposure to low doses of ionizing radiation?
Terminal deletions Single hit kinetics predominates at low doses of ionizing radiation. Terminal deletions involve a single double-strand break, and therefore follow single-hit kinetics.
The MRN complex
The NBS/MRE11/Rad50 complex is involved in "end processing," while Ku70, Ku80, and DNA-PKcs are involved in "end recognition."
The minimum whole body radiation dose that can be detected through the measurement of dicentric chromosomes in peripheral blood lymphocytes is approximately:
The minimum whole body dose that can be detected through measurement of dicentric chromosomes in peripheral blood lymphocytes is approximately 0.25 Gy.
Most reactive oxygen species in a cell are produced from mitochondrial respiration.
The neutral comet assay is used to measure DNA double-strand breaks. Alkaline elution is used to measure single-strand breaks and some base damages, western blotting is for detection of proteins, PCR (polymerase chain reaction) is used to amplify DNA sequences, and annexin V-labeling can be used to detect apoptosis.
How does the slope of the survival curve change with more potentially-lethal damage (PLD) repair?
The slope decreases because cell survival is increased through the action of PLD repair.
Holliday junction resolution is a component of homologous recombination. T/F
True Holliday junctions are formed following the invasion of the homologous strand of the sister chromatid in homologous recombination repair.
Terminal deletions are induced as a linear function of dose T/F
True since they result from a single chromosomal break.
Which of the following processes involves Large numbers of complex rearrangements in localized regions of single chromosomes
chromothripsis Chromothripsis is the phenomenon by which up to thousands of clustered chromosomal rearrangements occur in a single event in localized and confined genomic regions in one or a few chromosomes and is known to be involved in both cancer and congenital diseases. It occurs through one massive genomic rearrangement during a single catastrophic event in the cell's history.
What type of chromosomal aberrations is detectable by CGH (comparative genomic hybridization)?
deletion CGH is only able to detect unbalanced chromosomal abnormalities. This is because balanced chromosomal abnormalities such as reciprocal translocations, inversions or ring chromosomes do not affect copy number, which is what is detected by CGH technologies.
PLD is repaired when survival is higher in:
density-inhibited cells that are subcultured 12 hours after irradiation compared to those plated immediately after irradiation. Potentially lethal damage (PLD) is said to be repaired when the subculture of non-cycling, density-inhibited cells is delayed, and the delay results in increased cell survival for what is otherwise the same radiation dose. Repair that is manifest as increased cell survival between fractionated doses is termed sublethal damage (SLD) repair.
Regarding cell survival, sublethal damage repair is:
described by the β component of LQ model. The shoulder region of the cell survival curve represents sublethal damage repair, and that is related to the beta component of cell kill. SLDR is generally complete by 1-2 hours.
In mammalian cells, sublethal radiation damage recovery is most likely due to the biochemical repair of DNA:
double-strand breaks.
Radiation-induced anaphase bridges generally result from:
isochromatid breaks
The majority of mutations in the ATM protein are _____.
truncating About 70% of the various mutations known in ATM are protein truncating in nature, whereas the remaining are missense mutations and short deletions or insertions, resulting in unstable ATM variants.
What is considered a biomarker of ionizing radiation-induced DNA double strand breaks?
γ-H2Ax
The majority of ionizing radiation-induced DNA DSBs typically are repaired in cells within the following timeframe:
2 - 24 hours
All of the following (ATM, ATR, RAD3, BRCA1 and DNA-PK) are phosphatidyl inositol 3-kinase like kinases, 𝐄𝐗𝐂𝐄𝐏𝐓
BRCA1 Phosphatidylinositol 3-kinase-related kinases (PIKKs) are a family of Ser/Thr-protein kinases with sequence similarity to phosphatidylinositol-3 kinases (PI3Ks). BRCA1 is not a phosphatidyl inositol 3-kinase, whereas ATM, ATR, RAD3 and DNA-PK all fall into this category of protein.
Increased numbers of chromosome aberrations, especially quadriradials, are frequently found even in the absence of radiation, in which of the following human syndromes?
Blood cells from individuals with Fanconi anemia are often found to have high numbers of chromosome aberrations, especially quadriradials. These complex aberrations increase dramatically with exposure to DNA cross-linking agents such as mitomycin c. Bloom's syndrome also has quadriradials.
how's anaphase bridge produced?
Breaks in two chromatids, followed by illegitimate rejoining, produce an anaphase bridge. They are formed after DNA synthesis, hence chromatid aberration.
Which protein is DIRECTLY involved in rejoining double-strand breaks by non-homologous end joining (NHEJ)?
DNA Ligase IV complexes with XRCC4 to join the two "broken" ends of a double-strand break.
During DNA single strand break repair, which step occurs first?
End processing by polynucleotide kinase End processing by polynucleotide kinase occurs before gap filling by DNA polymerase β
The formation of dicentric chromosome aberrations follows a linear-quadratic dose response curve. This has been interpreted to mean that the production of dicentric chromosomes results from:
Two chromosome breaks, produced either by one or by two separate radiation tracks the formation of dicentric chromosomes is linear at low radiation doses, but follows a quadratic function at higher doses. Two distinct mechanisms are thought to be responsible for these two components of the linear-quadratic dose response curve. The linear portion of the dose response relationship is assumed to result from the simultaneous induction of two chromosome breaks by a single track. The quadratic portion is assumed to result from the two chromosome breaks being produced by two separate radiation tracks.
Two-hit aberrations:
account for the β component of dose response curves. Two-hit aberrations (or DNA breaks) are dependent on the square of the dose; hence they are the β (quadratic) component of the doe response curve and will decrease with dose fractionation or decreasing dose rate, both of which allow time for repair.
