ASTRO RadBio 2017-XXXX Qs - DNA Repair Mechanisms

Which of the following is NOT a known substrate for ATM? a. Ku70/80 (XRCC6/XRCC5) b. BRCA1 c. NBS1 d. p53 (TP53) e. CHK2 (CHEK2)

A. All of the proteins listed are substrates for ATM except for Ku70/80.

Which of the following statements is correct? Base excision repair (BER): A. When defective, may increase mutation rate, but usually does not dramatically alter cellular radiosensitivity B. Is the principal pathway responsible for the repair of UV-induced DNA damages C. Involves the XP and CS genes D. Acts primarily on bulky DNA lesions induced by polycyclic aromatic hydrocarbons E. Is defective in patients with Li-Fraumeni Syndrome

A. Defects in base excision repair may increase mutation rate, but generally do not alter cell survival after ionizing radiation, with the exception of mutation in the XRCC1 gene which confers a slight increase in radiation sensitivity. Defects in nucleotide excision repair (NER) increase sensitivity to UV, but not to ionizing radiation. The xeroderma pigmentosum (XP) and Cockayne Syndrome (CS) genes are involved in NER. BER acts to remove damaged bases from DNA, including those damaged by ionizing radiation, but NER acts on pyrimidine dimers, single-strand breaks and bulky adducts. The gene defective in most patients with Li-Fraumeni Syndrome is p53, although some patients with that condition have mutations in CHK2.

Which of the following statements is FALSE? A. DNA repair by homologous recombination occurs preferentially in the G1 phase of the cell cycle B. Non-homologous end joining is an error-prone repair pathway that involves DNA-PKcs (PRKDC)-associated repair of DNA double-strand breaks C. The DNA repair proteins MRE11, NBS1 (NBN) and RAD50, localize at nuclear foci corresponding to presumed sites of DNA damage following exposure to DNA-damaging agents D. A defect in nucleotide excision repair is the basis for the hereditary disorder xeroderma pigmentosum and can lead to increased rates of skin cancer E. Following the production of DNA double-strand breaks, ATM is converted from an inactive dimer to an active monomer form

A. Homologous recombination requires a second copy of the relevant DNA duplex. Although homologous recombination can take place in G1 phase, using the homologous chromosome as the template for repair, it occurs much more frequently after replication when the template strand is the sister chromatid, located in close proximity to the damaged strand. The sister chromatid is created during S-phase and serves as a template from which to copy the intact DNA sequence to the site of the damaged strand of DNA. It has been estimated that homologous recombination occurs 1000-fold more frequently in S and G2 than in G1. In G1, the principal form of DNA double-strand break repair is non-homologous recombination

15. Which of the following is first involved in the process of non-homologous end joining? a. KU70 b. LIG4 c. DNA-PKcs d. BRCA2 e. ARTEMIS

A. KU70 binding is involved in the initial recognition of a DSB and is one of the first steps of NHEJ repair, followed by DNA-PKcs activation and binding to the ends. LIG4 is involved in sealing the ends by ligation in the NHEJ repair process. ARTEMIS has exonuclease activity and is also involved in NHEJ repair. BRCA2 is an HR protein and may have some NHEJ functions.

Two of the main proteins involved in mismatch repair are: A. MSH2/MLH1 B. DNA ligase IV (LIG4)/XRCC4 C. KU70 (XRCC6)/KU80 (XRCC5) D. XPA/XPG (ERCC5) E. DNA-PKcs (PRKDC)/Artemis

A. MSH2 and MLH1 play a central role in mismatch repair. Regarding the other proteins, XPA/XPC are involved in nucleotide excision repair and the remainder play roles in NHEJ.

Cells derived from individuals diagnosed with xeroderma pigmentosum are deficient in: a. Nucleotide excision repair b. Methyl-guanine transferase c. Mismatch repair d. Base excision repair e. Homologous recombination

A. People with xeroderma pigmentosum are deficient in one of the several proteins involved in nucleotide excision repair. They are therefore extremely sensitive to UV irradiation because they are unable to repair the pyrimidine dimers produced in DNA, but they are not sensitive to ionizing radiation.

Which of the following proteins is most involved in homologous recombinational repair of radiation-induced DNA double-strand breaks? A. RAD51 B. XPG (ERCC5) C. DNA-PKcs (PRKDC) D. CHK1 (CHEK1) E. TFIIH

A. RAD51 is a recombinase and plays a critical role in homologous recombinational repair of DNA double-strand breaks. XPG is an endonuclease that cleaves the DNA strand on the 3' side of the damage site; it also stabilizes the nucleotide excision repair pre-incision complex that is essential for the 5' incision by the XPF (ERCC4) endonuclease. The catalytic unit of DNA protein kinase (DNA-PKcs) plays a central role in non-homologous end joining of DNA double-strand breaks through its recruitment by the KU70 (XRCC6)/80 (XRCC5) heterodimer to sites of DNA double-strand breaks, forming the DNA-dependent protein kinase holo-enzyme complex (DNA-PK). CHK1 is a serine/threonine protein kinase and a key mediator of the DNA damage-induced checkpoint pathway. TFIIH is associated with nucleotide excision repair.

Normal tissues from which of the following syndromes shows the highest level of sensitivity to ionizing radiation? a. Ataxia telangiectasia b. Systemic lupus erythematosus c. Bloom's syndrome d. Xeroderma pigmentosum e. Fanconi's anemia

A. Radiation sensitivity is greatest in a person with ataxia telangiectasia. People with this syndrome are very sensitive to ionizing radiation due to the absence of functional ATM protein, which plays a central role in the repair of DNA double strand breaks and regulation of the cell cycle following irradiation.

8. All of the following statements about non-homologous end joining (NHEJ) are true, EXCEPT: 12 a. Artemis is primarily responsible for ligating broken DNA ends b. DNA ligase IV forms a tight complex with XRCC4 c. DNA-PKcs associates with Ku70/80 to form the DNA-PK holo-enzyme d. The Ku heterodimer has a high affinity for DNA ends and forms a close-fitting asymmetrical ring that threads onto a free end of DNA e. NHEJ is an error-prone process

A. The main role for Artemis is to cleave (through its nuclease activity) any residual DNA loops or hairpins that form during non-homologous end-joining. It prepares the ends for ligation by ligase.

Which syndrome is caused by a deficiency in the repair-associated protein MRE11? a. Werner's syndrome b. Ataxia-telangiectasia-like disorder c. Xeroderma pigmentosum d. Bloom's syndrome e. Cockayne's syndrome

B. A deficiency in MRE11 (which makes up the MRE11-RAD50-NBS1 complex) results in an ataxia telangiectasia-like disorder. The protein plays a role in both homologous recombination and NHEJ.

Which of the following proteins localizes to and is used as a marker of DNA double strand breaks? a. p53 b. Chk1 c. yH2AX d. Rb e. ATM

C. γH2AX, the phosphorylated histone variant H2AX, localizes to sites of DNA double strand breaks rapidly after damage and acts as a docking site for other DNA repair proteins.

Which of the following reflects a poor repair of DNA double strand breaks (DSB) after irradiation? a. Phosphorylation of H2AX 15 minutes after irradiation b. Phosphorylation of H2AX 24 hours after irradiation c. Persistent arrest at G1 phase 4 hours after irradiation d. Phosphorylation of ATM within 15 minutes after irradiation e. Phosphorylation of ATM within 24 hours after irradiation

B. Excessive level of γH2AX is an indication of weak DSB repair. The initial activation of ATM and γH2AX formation are normal cell responses to irradiation. Radiation induced G1 cell cycle arrest usually last for a few hours in human cells, thus the arrest in G1 after 4 hours after irradiation is considered a normal DNA damage response.

DNA double strand break repair occurs through several mechanisms. Defects in these pathways may alter cellular radiosensitivity. In mammalian cells, relative to homologous recombination (HR), defects in non- homologous end joining (NHEJ) are likely to lead to: a. Decreased radiosensitivity. b. Increased radiosensitivity c. Decreased radiosensitivity in quiescent cells only d. No difference in radiosensitivity in quiescent cells only e. No difference in radiosensitivity in dividing cells

B. In mammalian cells, NHEJ defects confer a higher sensitivity to radiation than HR. It contributes to sensitivity to both proliferative and quiescent cells

Double-strand DNA breaks caused by ionizing radiation trigger the transcription of DNA damage response genes. Which of the following proteins is a transcriptional transactivator? A. p21 (CDKN1A) B. p53 (TP53) C. ATM D. CHK1 (CHEK1) E. TRAIL (TNFSF10)

B. In response to various forms of DNA damage including double-strand breaks, p53 is stabilized and binds to the promoters of numerous target genes, including p21, activating their transcription. This transcriptional transactivation by p53 is an important component of the cellular DNA damage response. ATM and CHK1 are protein kinases that are activated in response to double-strand breaks. TRAIL is a ligand that induces cell death through the extrinsic apoptosis pathway.

Repair of DNA double-strand breaks can be accomplished by which one of the following pathways? a. Mismatch repair b. Non-homologous end joining c. Base excision repair d. Nucleotide excision repair e. Photoreactivation

B. Non-homologous end-joining represents the principal means by which human cells repair DNA double strand breaks. Mismatch repair is primarily responsible for correction of errors made during DNA replication. Base excision repair removes base damages. Nucleotide excision repair mainly removes bulky adducts from DNA such as UV-induced pyrimidine dimers and chemical adducts. Photoreactivation involves the action of DNA photolyase which is activated by long wavelength UV and visible light to split the cyclobutyl bond of a pyrimidine dimer restoring it back to its original state.

Which of the following is the most proximal signaling event in the DNA damage response in cells? a. BRCA2 loading onto a double-strand break (DSB) b. Gamma-H2AX phosphorylation c. DNA-PK catalytic subunit autophosphorylation d. BRCA1 recruitment via RNF8 to a DSB e. CtIP-induced resection of DSB ends

B. One of the first events that occurs at the site of an induced DSB is phosphorylation of the histone variant, H2AX at serine 139. ATM is the primary kinase, which phosphorylates H2AX, although ATR and DNA-PKcs also perform this action. This leads eventually to BRCA1 recruitment which likely is followed by CtIP binding and resection. NHEJ repair is initiated by DNA-PKcs, but likely occurs after H2AX phosphorylation.

Which of the following statements concerning DNA repair is CORRECT? A. Cells deficient in nucleotide excision repair tend to display hypersensitivity to ionizing radiation B. A person with LIG4 syndrome is radiation sensitive C. Mismatch repair involves the action of a DNA glycosylase and an AP endonuclease D. People with Fanconi anemia exhibit normal sensitivity to DNA cross-linking agents E. A mutation in p53 (TP53) produces an immune deficient phenotype in SCID mice

B. People diagnosed with LIG4 syndrome are radiation sensitive because these individuals are deficient in the DNA ligase IV enzyme (LIG4), which plays a central role in non-homologous end joining of double-strand breaks. Cells deficient in nucleotide excision repair exhibit normal sensitivity to ionizing radiation since this repair process plays little or no role in the repair of damages induced by ionizing radiation, but are very sensitive to UV. 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 immune deficient phenotype in SCID mice is caused by a defect in XRCC7 (DNA-PKcs); this defect causes a defect in NHEJ and a radiosensitive phenotype, as well as the defect in V(D)J rejoining that leads to the immune deficit. Defects in several genes are now known to cause SCID phenotypes; mutation in the common human disease of the same name differs from that in the well-known mouse strain.

All of the following proteins are involved in non-homologous end-joining of DNA double-strand breaks, EXCEPT: A. XRCC4 B. RAD52 C. Artemis (DCLRE1C) D. KU70 (XRCC6)/KU80 (XRCC5) E. DNA ligase IV (LIG4)

B. RAD52 plays a central role in homologous recombinational repair (HR) of DNA double-strand breaks through recruitment of RAD51 to single-stranded DNA complexed with RPA. RAD52 does not appear to be involved in NHEJ. XRCC4 is an adaptor protein that tightly complexes with DNA ligase IV, which directly mediates DNA-strand joining by NHEJ. The KU70/KU80 heterodimer recruits DNAPKcs (PRKDC) to the site of DNA double-strand breaks to form a multiprotein complex that keeps broken DNA ends in close proximity and provides a platform for the enzymes required for end processing and ligation. DNA-PKcs phosphorylates the Artemis protein, thereby activating it for endonucleolytic activity. The Artemis:DNA-PKcs complex cleaves 5´ and 3´ nucleotide overhangs, which prepares double-strand breaks for ligation by XRCC4 and DNA ligase IV.

All the following statements are true concerning homologous recombinational repair of DNA double strand breaks, EXCEPT: a. H2AX phosphorylation represents an important step in the formation of repair foci b. The BLM protein serves to coat single stranded DNA regions to prevent their degradation c. The MRN complex relocates to sites of DNA double strand breaks to process DNA resulting in production of single stranded ends d. RAD51 is a recombinase and forms a nucleoprotein filament that facilitates strand invasion for homologous recombination e. ATM is activated following irradiation by auto-phosphorylation and conversion from an inactive dimer to an active monomer

B. The BLM protein (deficient in people with Bloom Syndrome) is a RecQ helicase that works in the 3' to 5' direction. RPA (Replication Protein A) serves to coat single stranded DNA regions generated during homologous recombination, DNA replication, and other processes to prevent their degradation.

Non-homologous end joining is thought to be most active in which phase of the cell cycle? a. G0-phase b. G1-Phase c. G2-phase d. S-Phase e. Mitosis

B. The NHEJ pathway is considered more error prone than HR and occurs more frequently in cells. NHEJ is the predominant pathway in the G0/G1-phases of the cell cycle, when a sister chromatid is not available for HR repair. NHEJ is active other parts of the cell cycle, but its highest activity appears to be in G1. Recent studies suggest it may also be the predominant pathway in the G2/M-phases of the cell cycle after compaction of the chromatin, but further studies are needed to clarify this.

The following statement is true regarding BRCA1 and BRCA2: A. BRCA1 and BRCA2 mutations account for only a few cases of familial hereditary breast and ovarian cancer B. BRCA1-deficient cells are resistant to the DNA crosslinking agent mitomycin C C. The prevalence of BRCA1 mutation is higher than that of BRCA2 mutations D. BRCA1 and BRCA2 predominantly regulate homologous recombination as opposed to non-homologous end joining E. The breast cancer risks for carriers of BRCA1 and BRCA2 mutations are similar, but with later age of disease onset for the BRCA1 mutation

D. BRCA1 and BRCA2 predominantly regulate homologous recombination as opposed to non-homologous end joining

Which of the following proteins is NOT directly involved in repairing DNA double strand breaks? a. Artemis b. RAD51 c. DNA-PKcs d. CDK4 e. BRCA1

D. CDK4 is a cyclin dependent kinase that plays an important role in the progression of cells through G1 and into S phase. Artemis and DNA-PKcs play important roles in non-homologous end-joining of DNA double strand breaks, whereas RAD51 and BRCA1 are involved in the repair of double strand breaks through homologous recombination

What are the 2 most common DNA double-strand break repair pathways in mammalian cells? a. Single-strand annealing (SSA) and Homologous Recombination (HR) b. Mismatch repair (MMR) and Nucleotide Excision Repair (NER) c. Non-homologous end joining (NHEJ) and Microhomology mediated end joining (MMEJ) d. Non-homologous end joining (NHEJ) and Homologous Recombination (HR) e. Base excision repair (BER) and Homologous Recombination (HR)

D. DSBs are considered the most serious form of DNA damage, since they eventually lead to cell death if not properly repaired. Thus, complex systems have evolved to rapidly detect and repair these lesions. HR and NHEJ represent the two major pathways by which DSBs are repaired, and cells derived from patients with DSB repair gene mutations are profoundly radiosensitive. While HR utilizes homologous DNA sequences as a template for repair, NHEJ simply processes and re-ligates the exposed DNA termini of DSBs. The NHEJ pathway is considered more error prone than HR and occurs more frequently in cells. NHEJ is the predominant pathway in the G0/G1-phases of the cell cycle, while HR increases during S/G2, when a sister chromatid becomes available as a template for repair

Homologous recombination is thought to be most active in which phase of the cell cycle? a. M-phase b. G0/G1-Phase c. G2-phase d. S-Phase e. Mitosis

D. HR is most active when a sister chromatid is available as a template for repair, which is in the late S- phase of the cell cycle. Sister chromatids are available in G2 and early M-phase as well, but the chromatin is compacted and may not be as suitable for HR repair.

Homologous recombinational repair of DNA double strand breaks is most likely to occur: a. In G0 b. In G1 c. In early S phase d. In late S phase e. Throughout the cell cycle

D. Homologous recombinational repair requires the presence of a homologous DNA template, and is therefore most likely to occur following DNA replication in late S phase (when a sister chromatid is available as a template) or G2 phases of the cell cycle.

An agent that inhibits non-homologous end joining (NHEJ) repair of radiationinduced DNA double-strand breaks might be expected to do all of the following, EXCEPT: A. Affect the immune response B. Sensitize cells to low dose rate irradiation C. Decrease normal tissue tolerance during fractionated radiotherapy D. Increase cellular radioresistance E. Inhibit sublethal damage recovery

D. Inhibition of non-homologous end joining would be expected to decrease cellular radioresistance. An effect on immune response would be anticipated because inhibition of NHEJ would affect V(D)J recombination, thereby affecting antigen recognition. Cells and tissues would be sensitized to low dose-rate irradiation since the recovery that occurs at low dose-rates depends, at least in part, upon repair of double-strand breaks by NHEJ. Normal tissue tolerance doses would likely decrease due to radiosensitization. Sublethal damage recovery would be inhibited since this process depends, at least in part, on the repair of double-strand breaks.

RAD51 and BRCA2 function together: a. As inhibitors of cyclin dependent kinases b. To phosphorylate H2AX and NBS1 c. To enhance apoptosis by inhibiting p53 (TP53) d. In the initial steps of homologous recombination e. To play a central role in nucleotide excision repair

D. RAD51 and BRCA2 function together in homologous recombinational repair of DNA double strand breaks.

Which of the following statements is TRUE concerning DNA repair processes? a. Between 10-20% of the population is thought to be heterozygous for the types of mutations that are responsible for causing ataxia telangiectasia (AT) b. Non-homologous end-joining requires the involvement of a sister chromatid c. Mutations in the genes that encode proteins involved in translesion DNA synthesis are typically present in people who develop hereditary non-polyposis colon cancer d. The most common types of DNA damage induced by ionizing radiation are repaired through base excision repair e. Sublethal damage repair is significant for both x-rays and neutrons

D. The most common alterations produced in the DNA by radiation are base damages, which are repaired by base excision repair, a repair process that is usually rapid and accurate. The proportion of the population that is heterozygous for the types of mutations that are found in people with AT - typically protein truncation mutations - is roughly 1-2%. Non-homologous end joining does not require a sister chromatid. Mutation of the genes involved with mismatch repair, primarily MSH2 and MLH1, are often present in people who develop hereditary non-polyposis colon cancer. Homologous recombination is a relatively error- free process. Sublethal damage repair is nearly non-existent following neutron-irradiation.

Which statement regarding the roles of non-homologous end-joining (NHEJ) and homologous recombination (HR) in the repair of ionizing radiation-induced DNA double-strand breaks (DSBs) is TRUE? A. HR removes DSB from the genome at a faster rate than NHEJ. B. Defects in HR compromise DSB repair, but do not affect the repair of damage at DNA replication forks. C. NHEJ requires homologies of 200-600 nucleotides between broken ends of DNA. D. Defects in NHEJ increase radiosensitivity more than defects in HR in mammalian cells.

D. Two principal recombinational DNA repair pathways have been identified, homologous recombination (HR) and non-homologous end-joining (NHEJ), each of which employs separate protein complexes. DSB repair by HR requires an undamaged template molecule that contains a homologous DNA sequence, typically derived from the sister chromatid in the S and G2 phase cells. In contrast, NHEJ of double-stranded DNA ends, which can occur in any cell-cycle phase, does not require an undamaged partner and does not rely on extensive homologies between the recombining ends (typically 2-6 bp of microhomology are used). Defective HR can be causally linked to impaired DNA replication, genomic instability, human chromosomal instability syndromes, cancer development, and cellular hypersensitivity to DNA damaging agents. Cells with genetic defects in NHEJ (such as mutation of DNA-PK, XRCC4, or DNA ligase IV) display a more pronounced hypersensitivity to ionizing radiation than cells defective in HR (such as mutation of BRCA1, BRCA2, or RAD51).

Which of the following molecular events occurs earlier than others following the creation of a double-strand DNA break? A. Destabilization of the mitochondrial outer membrane B. Inactivation of the CDC25 phosphatases C. Phosphorylation of CHK1 (CHEK1) D. Activation of p21 (CDKN1A) transcription E. Phosphorylation of histone H2AX

E . Phosphorylation of histone H2AX to -H2AX occurs within several minutes of a cell being irradiated. This modification is triggered by ATM and serves to mark the chromosomal site of the DNA break for the subsequent recruitment of signaling proteins, such as CHK1 kinase. Activated CHK1 phosphorylates and inactivates CDC25 proteins, thereby causing the arrest of the cell cycle. p21 transcription is induced several hours after DNA damage, following the stabilization of p53 (TP53).

Which of the following best describes the action of an exonuclease enzyme? A. Seals breaks in a DNA strand B. Required for DNA replication C. Produces nicks within intact DNA strands D. Controls mRNA synthesis E. Removes nucleotides from the ends of DNA strands

E. An exonuclease cleaves one nucleotides at a time beginning at the end of a DNA strand.

SCID mice are often used in cancer research because they: a. Are radioresistant b. Exhibit high levels of non-homologous end joining c. Have efficient immune systems d. Are better able to repair radiation damage e. Are useful hosts for growing human tumor xenografts

E. SCID mice are immune deficient, making them good hosts for growing xenografts of human tumors. SCID mice are deficient in DNA-PK and are therefore radiosensitive. Cells from these mice have low levels of non- homologous end-joining. Nevertheless, it is their lack of an immune system that permits the human tumor cells to survive in a mouse without being rejected.

A mutation in which of the following genes is LEAST likely to cause an increase in sensitivity to ionizing radiation: A. NBS1(NBN) B. BRCA1 C. ATM D. MRE11 E. XPC

E. XPC is a gene whose product is involved in nucleotide excision repair. Mutations in XPC result in the human genetic disease xeroderma pigmentosum, characterized by extreme sensitivity to ultraviolet light. Mutations in all of the other genes result in human genetic diseases characterized by sensitivity to ionizing radiation. This includes Nijmegen breakage syndrome (NBS1), familial breast cancer (BRCA1), ataxia telangiectasia (ATM), and ataxia telangiectasia-like disorder (MRE11).