Nepveu Lectures (Repair Lectures 1-4) Flashcards

Question

What is the model for double-stranded break repair?

Answer 1

(A) Recombination repair pathway (homology-dependent repair) (HR) - involves nucleolytic resection of one strand followed by strand invasion of a homologous DNA molecule (sister chromatic and DNA repair synthesis). - sister chromatic is used as a template to replace the genetic information lost during the nucleolytic process. (B) Non-homologous end-joining pathway (NHEJ) - Kv heterodimer binds to DNA ends and recruits DNA-PKcs. - may be repaired accurately or inaccurately. - goes through re-ligation or fill-in/deletion ligation.

Answer 2

- key idea is that a DSB in DNA is a trigger for homologous recombination. 1) Circular plasmid. 2) Linearized plasmid. 3) Linearized plasmid with a deletion.

Answer 3

(1) Recognition of double-stranded break. - MRN complex recruited to break, binds to DNA through MRE11. - RAD50, ATM, NBS1, BRCA1, 4H2AX, MDL1. - RNF8 -> initiated ubiquitylation cascade of histones H2A & H2AX causing chromatin restruction and generation of binding sites for further protein factors. (2) End-processing: - damaged DNA ends are processed to prepare for repair. - CtIP starts process by performing 5' resection. - this relies on CtIps recruitment of BRCA1. - more resection carried out by EXO1, producing SS-DNA with 3' overhangs. (3) Nucleoprotein filament formation: - RAD51 monomers bind to ss-DNA 3' overhangs, forming RAD51 nucleoprotein filaments. (4) RAD51 recombinase complex assembly. - built with accessory proteins (BRCA 1/2, RAD52, RAD51 paralogs) (5) Homology search, strand invasion & D-loop formation. - Rad51 recombinase complex & RAD54 search for homologous DNA sequence in sister chromatid/homologous chromosome. - strand invasion occurs, forming D-loop. (6) DNA repair synthesis. - DNA polymerase uses homologous strand in D-loop as template to extend 3' invading strand through DNA repair synthesis. -> branch migration -> holiday junction. -> capture of second DNA end -> double holiday junction (DHJ). - LIG1 seals - structure-specific endonuclease cut junctions to produce either: - cross-over products (swapped DNA regions). - non-cross over products. - the BLM RecQ helicase with TOPO3a & BLAP65, can make non-crossover products.

Answer 4

(1) Recognition of double-stranded break. - MRN complex recruited to break, binds to DNA through MRE11. - RAD50, ATM, NBS1, BRCA1, 4H2AX, MDL1. - RNF8 -> initiated ubiquitylation cascade of histones H2A & H2AX causing chromatin restruction and generation of binding sites for further protein factors. (2) End binding. - Ku70-Ku80 heterodimer binds to broken DNA ends. (protects DNA termini) (3) DNA-PK complex formation. - DNA-PKCS recruited to DNA ends by Ku70-Ku80. - Forms the DNA-PK complex, holds two broken DNA ends close together. (4) End processing and resection. - DNA-PKCS autophosphorylates. - Processing enzymes TDP1, FEN1, WRN helicase, artemis. - Produce 5'-P & 3'-OH termini required for ligation. (5) DNA-repair synthesis. - DNA polymerase fill in any missing nucleotides. - DNA-PK then recruits LIG4-XRCC4-XCF complex. - LIG4 seals DNA ends (ligation).

Answer 5

Because lesions that persist hamper the replication apparatus, cells have evolved a damage tolerance system to allow complete replication in the presence of DNA damage. This response tolerates, rather than removes, DNA damage, and consists of at least two mechanisms.

Answer 6

1) Template switching. 2) Lesion Bypass (translesion synthesis).

Answer 7

a) high levels of damage. b) poorly repaired lesions. c) inefficiently repaired genomic regions. d) DNA damage occurred during the S phase of the cell cycle.

Answer 8

"post-replication repair" - avoids replication of the damaged site of the DNA template. - by using the newly synthesized daughter strand as template (template switching), the replicative machinery effectively circumvents the lesion block DNA and proceeds with replication.

Answer 9

- directly utilizes the damaged template. - can be divided into two steps: i) nucleotide incorporation opposite the lesion (i.e., translesion synthesis), followed by ii) extension of DNA synthesis. After a short stretch of extension, normal DNA synthesis by the replication apparatus can then resume. Error-prone lesion bypass constitutes the major mechanism of DNA damage-induced mutagenesis in cells.

Answer 10

- specialized DNA polymerases (Pol) that can use damaged DNA as template: the Y family (UmuC superfamily): Umu for "umutable." *** lesion bypass can either be error-free or error-prone.

Answer 11

Completion of DNA replication.

Answer 12

- the gene names Umu (unmutable) in E.coli and Rev (reversion-negative) in yeast refer to the fact that mutants in these genes exhibit a reduced rate of mutations. - Indeed, most mutations in bacteria and yeast are caused by the action of translesion synthesis polymerase.

Answer 13

1) Binding of LexA repressor to regulatory operators upstream of SOS genes (limits their expression under normal growth conditions) 2) RecA induced from UV, activated by binding to regions of ssDNA to form nucleoprotein filament, RecA* 3) RecA* coprotease in autocleavage of LexA → induction of SOS genes. 4) Proteolytic processing event, UmuD, forms UmuD’, two form with one UmuC to form polV (2 UmuD’ + 1 C).

Answer 14

- Refers to a process whereby mis-adaption of bacteria to a new environmental conditions (lack of nutrients, change in temperature, antibiotics) triggers the induction of error-prone polymerases whose action increases mutation frequency). - This process produces a highly heterogenous population of genetic variants, increasing the probability of obtaining a clone that can proliferate in new conditions. - This process does not exist in higher organisms, although an analogous process takes place during somatic hypermutation in B cells. (somatic mutation is one example where specialized cells purposely increase the frequency of mutations in a specific genomic locus)

Answer 15

1) Purpose of SHM: SHM generates mutations in immunoglobulin genes to create antibodies with higher affinity for antigens. 2) Initiation by AID: Activation-induced cytidine deaminase (AID, yellow oval) initiates SHM. AID deaminates cytosine (C) in DNA, converting it to uracil (U) (red). 3) Role of Transcription: RNA polymerase II (RNA Pol II, brown oval) exposes single-stranded DNA during transcription, providing access for AID. 4) Mutations at C-G Pairs: --> Replication over uracil: Replicating over uracil causes C → T or G → A mutations (transition mutations). --> Error-prone repair by Ung: Uracil DNA glycosylase (Ung, blue oval) removes the uracil, leaving an abasic site (F). Error-prone repair by translesion synthesis polymerases (Rev1, yellow square, or TLS pol, purple diamond) can cause transition or transversion mutations (labeled as ‘N’). 5) Mutations at A-T Pairs: --> Mismatch recognition by Msh2/Msh6: The U-G mismatch is recognized by mismatch repair proteins Msh2/Msh6 (green and purple ovals). These recruit exonuclease 1 (Exo1, yellow triangle) and polymerase eta (Polh, pink oval), spreading mutations to nearby A-T base pairs (also labeled as ‘N’). 6) High-Fidelity Repair: --> BER and MMR pathways: Ung and Msh2/Msh6 can function in normal base excision repair (BER) or mismatch repair (MMR), accurately repairing the uracil without introducing mutations.

Answer 16

- In a WT-strain, the serial action of base selectivity, proofreading and mismatch repair reduces the error rate of DNA replication to a low level. - In a mismatch-repair-defective phenotype (mutL), the error rates represent the efficiency of base selection and proofreading. - In a double-mutant for mismatch repair and proofreading (mutDmutL), the error rates represent the efficiency of base selectivity only.

Answer 17

- the up-regulation of PollV and PolV. - the inhibition of mismatch repair.

Answer 18

You would use BER. DNA glycosylases. APE1. PolB. PARP1. XRCC1. LIG3 or long patch repair: DNA pol epislon/delta LIG1.

Answer 19

HDR: - MRN complex - Ctlp - RAD51 complex (RAD51/BRCA1/BRCA2/RAD52) NHEJ: -MRN complex - Ku70 -KU80 -DNA-PK -WRN-RecQ

Answer 20

NER 1) GG-NER: XPC XPE XPB XPD RPA XPA XPF-ERCC1 XPG DNA Pol epsilon/delta/k LIG1 2) TC-NER: CSA CSB

Answer 21

Mismatch repair E.coli: MutS MutL MutH Mammals: MSH2/MSH6 MSH2/MSH3 MLH1-PMS2 Exo1 DNA Pol epsilon LIG1

Answer 22

Senses the DNA damage and initiates kinases, which then will initiate effector kinases. Initiating Kinases: ATM ATR DNA-PK Effector Kinases CHK1 (by ATM) CHK2 (by ATR)

Answer 23

Ataxia-telangiectasia (AT) is a human autosomal recessive genetic disorder which is characterized by symptoms including immune deficiencies, neuronal degeneration, growth retardation, and an approximate 100-fold increase in the incidence of cancers.

Answer 24

Like ATM, ATR is a nuclear protein kinase. The protein kinase activity of the ATR/Rad3 (MEC1) homologues is essential for mediating checkpoint regulation.

Answer 25

Mice that carry inactivating mutations in either Ku70, Ku80 or DNA-PK are defective in DSB repair. These mutants are hypersensitive to ionizing radiation, defective in V(D)J recombination and suffer from severe combined immunodeficiency (SCID).

Answer 26

- multiple types of post-translational modifications and complex signalling cascades.

Answer 27

(1) Sumolyation of PCNA prevents recombination during replication. (2) Mono-ubiquitination leads to translesion synthesis, and poly-ubiquitination promotes template switching.

Answer 28

Both checkpoints involve the activation of the ATM and ATR kinases, which phosphorylate and activate the checkpoint kinases, CHK1 and CHK2. These kinases phosphorylate and inactivate the G1/S and G2/M phosphotases, Cdc25A and Cdc25C. In the absence of these phosphotases, cyclin E/CDK2 and cyclin B/CDK1 remain phosphorylated and inactive. Another target of the ATM/ATR kinases in G1 is the p53 tumour suppressor. Phosphorylated p53 can activate transcription of the gene coding for CDK inhibitor, p21WAF/CIP1.

Answer 29

*checkpoint-induced degradation of the CDC25A phosphatase. After a DNA double-strand break (DSB) generation and ATM activation, the rate of phosphate incorporation into CDC25A increases through combined action of CHK1 and CHK2. This leads to stronger interaction with ubiquitin ligase, reduction of CDC25A half life, and S-phase delay.

Answer 30

(1) - During prophase/early prometaphase, kinetochore assembly recruits the spindle assembly checkpoint (SAC) complex to unattached kinetochores. - SAC generates a diffusible "wait anaphase" signal that inhibits/sequesters CDC20, an activator of the anaphase-promoting complex/cyclosome (APC/C). - Separase, the protease that cleaves cohesins holding sister chromatids, is inhibited by securin binding.

Answer 31

(3) Following silencing of the signaling at each kinetochore and turnover of the inhibitor that transmits the wait anaphase signal, APC/C-mediated ubiquitylation (Ub) of securin and cyclin B1 and subsequent degradation by the proteosome triggers anaphase entry.

Answer 32

(2) Methaphase As each pair of sister kinetochores attaches to kinetochore microtubules (MT, green), and microtubule motors generate tension that stretches them, generation of the checkpoint inhibitor is silenced at those kinetochores.

Answer 33

1) Tumour maintenance and tumour progression require that cancer cells surmount many obstacles and adapt to selective pressures of several types. 2) A single tumour cell with a static genome cannot successfully survive these multiplication steps. 3) "Success" in tumour maintenance and tumour progression necessitates that a tumour cell population will be large enough and evolves rapidly enough to be able to generate variants that will survive the next selection step.

Answer 34

The concept that cancer cells exhibit a mutator phenotype (mutate at a high rate) states that the large number of mutations in tumour cells cannot be accounted for by the low mutation rates of normal somatic cells, but instead must be a manifestation of a mutator phenotype. "Cancer cells have way more mutations than normal cells. Normal cells mutate at a very low rate, so there’s no way that all those cancer mutations could happen just by chance. Instead, cancer cells seem to have something special going on that makes them mutate a lot faster. This high mutation rate is called a mutator phenotype—basically, cancer cells are "mutation machines.""

Answer 35

- Aneuploidy - Copy number variations (chromosome loss, amplifications, deletions)

Answer 36

1) Oncogenes: Often missense mutations. 2) Tumour suppressor genes: More often nonsense and frameshift mutations.

Answer 37

(1) Cancer families: Exhibit a predisposition to develop certain types of cancer. Members of these families typically have inherited a germ-line mutation in a tumour suppressor gene (ex.BRCA1, BRCA2, HNPCC). (2) Somatic mutations: Acquired during the person's life.

Answer 38

When one allele is deleted such that only one allele remains. The remaining allele may or may not be inactivated by a point mutation, a short deletion or transcriptional silencing.

Answer 39

Stipulates that the two alleles of a tumor suppressor gene must be inactivated (by deletion, point mutation or silencing) to produce a cancerous phenotype. In contrast, in the case of haploinsufficient tumor suppressor genes, inactivation of only one allele is sufficient to confer a cancerous phenotype. In familial retinoblastoma, a person is born with one defective copy of the Rb gene in all cells. If the remaining normal copy gets mutated in a retinal cell, that cell loses Rb function and can develop into a tumor. In sporadic retinoblastoma, both copies of the Rb gene must be mutated in the same cell for a tumor to form. Since the chance of mutating one copy is already rare, the chance of mutating both is extremely low, making it unlikely to happen in many cells. This usually results in only one tumor.

Answer 40

(1) Intrinsic mutation process: - endogenous DNA damage - errors during DNA repair - translesion synthesis (2) Environmental & lifestyle exposure: - smoking - UV irradiation (farmers) (3) Mutator phenotype - inactivation of DNA repair - more ROS - Tetraploidy & chromosome - merotely - Chromothrypsis - Kategis - AID & APOBEC (4) Chemotherapy & radiotherapy therapy-related leukemia.

Answer 41

Microsatellite Instability (MSI) , observed in tumours which inactivating mutations in mismatch repair genes (ex. hMSH2, hMLH1). These tumour cells not only acquire more mutations in microsatellite sequences but also in coding sequences. Mice homozygous for KO mutations of the Msh2, MIh1, or Pms2 genes exhibit MSI and a predisposition to cancer.

Answer 42

A germline mutation in one allele of a mismatch repair gene. Tumours are formed from cells in which the remaining normal allele is inactivated.

Answer 43

(Greek for ‘‘shower’’ or ‘‘thunderstorm’’) a phenomenon whereby point mutations rapidly occur and cluster over hundreds (microcluster) or millions (macrocluster) of DNA bases.

Answer 44

a phenomenon whereby individual chromosomes are shattered and reassembled in a single catastrophic event. The mechanism for chromothripsis starts with the physical isolation of chromosomes in aberrant nuclear structures called micronuclei. This is followed with the fragmentation and subsequent reassembly of a single chromatid during the next cell division. Catastrophic chromosomal breakage -> attempted chromosomal repair -> progression towards cancer.

Answer 45

(1) Increasing antioxidant production to balance ROS levels and protect the cell from damage. (2) Speeding up the repair of oxidative DNA damage, allowing cells to avoid senescence (aging) and keep dividing, even in a high-ROS environment. Proteins like Sestrins help in this process by regenerating peroxiredoxins, which are crucial for reducing ROS and maintaining the cell's antioxidant defenses.

Answer 46

- Increase the frequency of mutations. - High ROS is another mechanism to generate a mutator phenotype.

Answer 47

- Tetraploid cells can form through: Cytokinesis failure Cell fusion Mitotic slippage Resulting in supernumerary centrosomes (extra centrosomes in the cell). -In normal cells: Extra centrosomes cause chaotic multipolar mitosis, often leading to non-viable daughter cells. -In some cancer cells: Extra centrosomes can be clustered into two poles, promoting bipolar mitosis. These cells overexpress spindle assembly checkpoint (SAC) proteins, giving them a strong SAC that extends mitosis. The robust SAC allows cells to successfully cluster centrosomes and divide, even with extra centrosomes. - Non-oncogene addiction: This adaptation is not due to reliance on a single oncogene, but rather the cancer cells' ability to adapt to the presence of extra centrosomes through mitotic regulation, specifically by manipulating the SAC and centrosome clustering. This is an example of mitotic adaptation rather than addiction to an oncogene.

Answer 48

- Multipolar spindle intermediates promote merotelic attachment. - Metastases may often derive from primary cancer cells that are teraploid.

Answer 49

- Therapy-related cancers are new cancers that develop after successful treatment for a primary cancer. - Cancer survivors have a significantly higher risk of developing a second primary cancer compared to the general population. - These cancers can arise due to various treatments, such as radiotherapy and alkylating agents, and the risk is often dose-dependent. Relative risk patterns vary for different therapies: 1) For Hodgkin lymphoma patients, the risk of developing acute myeloid leukemia (AML) peaks 2–7 years after treatment with chemotherapy. 2) Radiogenic breast cancer risk from radiotherapy peaks 15–20 years post-treatment and remains elevated thereafter. 3) Lung cancer risk after radiotherapy for Hodgkin lymphoma is dose-dependent and increases linearly with radiation dose.

Answer 50

- cancer treatment - ionizing radiations will cause oxidative DNA damage, including base damage, single-strand breaks and double-strand breaks.

Answer 51

All the agents used cause mutations in DNA (alkylating agents, topo inhibitor) or target DNA metabolism (antimetabolite) or chromosome segregation (antimicrotubule agents). - Antimicrotuble agents - Alkylating agents - Antimetabolites - Topoisomerase Inhibitors

Answer 52

- For example, glioblastoma cancer cells that express higher levels of MGMT, either because of gene amplification or overexpression, exhibit resistance to the alkylating agent, temozolomide.

Answer 53

The paradigm for "personalized medicine", also called precision medicine, was established through research on breast cancer. Only the HER2 breast cancer subtype responds to trastuzumab.

Answer 54

Two genes are synthetic lethal only when their simultaneous inactivation results in cellular or organismal death. In this example, deletion of either gene A or gene B does not affect viability whereas inactivation of both at the same time is lethal.

Answer 55

Have cancer cells and normal cells. 1) Infect with pools of lentiviruses expression shRNAs. 2) Quantify shRNAs in each cell population. 3) Genes synthetic lethal in cancer cell.

Answer 56

The synthetic lethality window (or therapeutic window) refers to the range of gene inhibition where mutant cells are selectively killed without affecting wild-type cells.

Answer 57

These include metabolic stress, proteotoxic stress, mitotic stress, oxidative stress, and DNA damage stress. These stresses generate additional pressures that select for extensive adaptations in cellular processes that are not themselves oncogenic. Yet, cancer cells are acutely dependent on heightened expression or activity of some normal proteins. Hence, the term: non-oncogene addiction.

Answer 58

The idea that cancer cells become unusually dependent on certain normal genes that are not themselves cancer-causing (not classical oncogenes). These genes are part of pathways that help cells cope with stress, and while healthy cells can survive without them, cancer cells rely on them to survive and grow in their stressful environment.

Answer 59

A practical application of this concept is that it offers additional therapeutic targets that could be exploited in cancer treatment. Therapeutics that interfere with the functions of “non-oncogenes” could display synthetic lethality with cancer cells.

Answer 60

- DNA lesions are typically repaired by multiple pathways that promote survival. - Synthetic lethality occurs when all repair pathways are blocked, leading to cell death. -Single-strand breaks (SSBs) are repaired by the PARP1-dependent pathway before replication forks arrive. - If SSBs are not repaired before the fork hits, they turn into double-strand breaks (DSEs). - DSEs at the replication fork are repaired by template switching, requiring BRCA1/2. - BRCA1/2-defective tumors cannot repair DSEs at collapsed forks, leading to dependency on PARP1 repair. - PARP inhibitors cause synthetic lethality in BRCA1/2-defective tumors because SSBs cannot be repaired in time, and DSEs cannot be fixed. - Normal tissue without bi-allelic BRCA1/2 defects is not affected by PARP inhibitors.

Nepveu Lectures (Repair Lectures 1-4) Flashcards

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