Molecular mechanisms of DNA Repair Flashcards
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
Transcriptional transactivators increase the expression of a protein by binding to the promoter region of the target gene and turning on transcription. 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 (Answer Choices C and D).
TRAIL is a ligand that induces cell death through the extrinsic apoptosis pathway (Answer Choice E).
Which of the following molecular events occurs earlier than the other events 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 gamma-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 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 doublestrand 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
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 (Answer Choice B).
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; Answer Choice C).
CHK1 is a serine/threonine protein kinase and a key mediator of the DNA damage-induced checkpoint pathway (Answer Choice D).
TFIIH is associated with nucleotide excision repair (Answer Choice E).
An agent that inhibits non-homologous end joining (NHEJ) repair of radiation-induced DNA double-strand breaks might be expected to do all of the following, EXCEPT:
A. Impact 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 (NHEJ) 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 (Answer Choice A).
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 (Answer Choice B).
Normal tissue tolerance doses would likely decrease due to radiosensitization (Answer Choice C).
Sublethal damage recovery would be inhibited since this process depends at least in part on the repair of double-strand breaks (Answer Choice E).
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 singlestranded 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 (Answer Choice A).
The KU70/KU80 heterodimer recruits DNA-PKcs (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 (Answer Choice D).
DNA-PKcs phosphorylate 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 (Answer Choice C and E).
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 (NER). Mutations in XPC result in the human genetic disease xeroderma pigmentosum, which is 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, including Nijmegen breakage syndrome (NBS1), familial breast cancer (BRCA1), ataxia telangiectasia (ATM), and ataxia telangiectasia-like disorder (MRE11).
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 crosslinking 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 (NHEJ) 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 radiation (Answer Choice A).
Base excision repair (BER), not mismatch repair, involves the action of a DNA glycosylase and an AP endonuclease (Answer Choice C).
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 (Answer Choice D).
The immune deficient phenotype in SCID mice is caused by a defect in XRCC7 (DNA-PKcs), which is critical for NHEJ as well as V(D)J rejoining. As a result, a defect in XRCC7 leads to a radiosensitive phenotype as well as the immune deficits seen in the SCID mouse. Defects in several genes are now known to cause SCID phenotypes; the mutation in the common human disease of the same name (severe combined immunodeficiency) differs from that in the well-known mouse strain.
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. XPA/XPG are involved in nucleotide excision repair (Answer Choice D). DNA Ligase IV, Ku70, and DNA-PKcs all play roles in NHEJ (Answer Choices B, C, and E).
Which of the following best describes the action of an exonuclease enzyme?
A. Seals breaks in a DNA strand
B. Adds a new nucleotide to the end of DNA during DNA synthesis.
C. Produces nicks within intact DNA strands
D. Generates new species of mRNA
E. Removes nucleotides from the ends of DNA strands
E
An exonuclease cleaves one nucleotide at a time beginning at the end of a DNA strand.
Ligases seal breaks in the DNA strand (Answer choice A). DNA polymerases add a new nucleotide to the end of DNA during DNA synthesis (Answer choice B). Endonucleases produce nicks within intact DNA strands (Answer choice C). RNA polymerase generates new species of mRNA (Answer choice D).
Which of the following statements is CORRECT? Base excision repair (BER):
A. May increase mutation rate when defective, but usually does not dramatically alter cellular radiosensitivity
B. Is the principal pathway responsible for the repair of UV-induced DNA damage
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 (BER) may increase mutation rate but generally do not alter cell survival after ionizing radiation with the exception of mutation of the XRCC1 gene, which would confer a slight increase in radiation sensitivity, as it is also involved in single-stranded DNA break repair.
Defects in nucleotide excision repair (NER) increase sensitivity to UV radiation but not to ionizing radiation (Answer Choice B).
The xeroderma pigmentosum (XP) and Cockayne Syndrome (CS) genes are involved in NER (Answer Choice C).
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 (Answer Choice D).
The gene defective in most patients with Li-Fraumeni Syndrome is p53, although some patients with that condition have mutations in CHK2 (Answer Choice E).
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 DSBs 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.
E. The cell cycle status does not affect the choice between HR and NHEJ to repair a DSB.
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).
Chemotherapeutic agents frequently produce DNA double-strand breaks (DSBs) by causing stalling and collapse of DNA replication forks. Which of the following pathways has a dominant role in the repair of replication associated double-strand breaks?
A. Non-homologous end-joining (NHEJ)
B. Homologous recombination (HR)
C. Single-strand annealing (SSA)
D. Translesional DNA synthesis (TLS)
E. Nucleotide excision repair (NER)
B
Several DNA repair pathways, including translesional DNA synthesis (TLS), nucleotide excision repair (NER), and homologous recombination (HR) can be mobilized at stalled DNA replication forks depending on the type of fork-blocking lesion. Chemotherapy-induced DNA lesions, such as interstrand crosslinks, interfere with the progress of the replicative DNA helicase or DNA polymerases, thereby leading to replication fork blockage or demise and producing DNA gaps or one-sided DNA double strand breaks (DSBs). Uncoupling of the replicative DNA helicase from the polymerases may occur generating excessive single-stranded DNA, which could in turn be the target of endonucleolytic processing, resulting in a one-sided DSB. In addition, single-stranded breaks induced by endogenous and exogenous sources may lead to the formation of one-sided DSBs due to runoff of the replication fork. In the repair of one-sided DSBs, HR appears to be the only pathway leading to their productive resolution. This entails resection of the DSB to form a 3′-tailed end for Rad51 filament assembly and DNA strand invasion and ultimately reconstruction of the replication fork.
A human disorder thought to be due to a DNA repair deficiency is which of the following:
A. Lesch-Nyhan syndrome
B. Xeroderma pigmentosum
C. Tay-Sachs disease
D. Phenylketonuria
E. Down syndrome
B
Xeroderma pigmentosum is a genetic disorder due to mutation in genes involved in nucleotide excision repair (NER). This phenotype is characterized by extreme sensitivity to ultraviolet light.
Which of the following statements 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 (HR) as opposed to non-homologous end joining (NHEJ).
Fewer than 10% of patients with breast cancer are found to have mutations in these breast cancer susceptibility genes. Due to the negative effect that BRCA1 mutation has on HR, BRCA1-deficient cells are more SENSITIVE, not resistant, to mitomycin C and other DNA crosslinking agents (Answer choice A).
The prevalence of BRCA1 mutation is slightly lower than that of BRCA2 mutations in the general US population (1:500 for BRCA1 vs 1:222 for BRCA2). The breast cancer risks for carriers of BRCA1 and BRCA2 mutations are similar at about 70% by the age of 80, but with earlier age of disease onset for the BRCA1 mutation (peaks in 4th decade) compared to BRCA2 carriers (peaks in 5th decade).
