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BIOCHEMISTRY 2 > Qiao2-3-Repair,Recombination > Flashcards

Qiao2-3-Repair,Recombination Flashcards

1
Q

What are the sources of DNA damage?

A
  1. Mistakes during replication
  2. Spontaneous mutation
  3. Induced mutations caused by environmental agents
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2
Q

What are some spontaneous DNA damage mechanisms?

A

1- Base loss/depurination: thousands of purines lost/day bc N-glycosyl linkage to deoxyribose is hydrolyzed -> AP (apurine/apyrimidine) site

2- Deamination of bases: deamination of cytosine to uracyl at ~100 bases/cell/day leads to C to T transition mutation. Also, conversion of adenine to hypoxanthine, and guanine to xanthine

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3
Q

What occurs with deaminations?

A
  • Spontaneous, pH and temp. dependent
  • May lead to mutations C->U, so will lead to C->T transition in next round of replication bc uracyl acts as thymine & pairs w/ adenine
  • Nitrous acid (HNO2) formed from nitrites in preserved meats react w/ stomach acid causing oxidative deamination
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4
Q

What is oxidative DNA damage?

A
  • Caused by ROS produced during mitochondria respiration & peroxide radicals
  • ROS introduces ~20K lesions/cell/day
  • Spontaneous modifications: hydrolytic attack, oxidative damage, uncontrolled methylation by SAM
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5
Q

What are the types of UV radiation?

A

UV-C (180-290nm): germicidal, most energetic & lethal. Absorbed by ozone layer

UV-B (290-320nm): major lethal/mutagenic fraction of sunlight

UV-A (320nm-visible): near UV, deleterious effects (bc it creates O-radicals), but it produces very few pyrimidine dimers

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6
Q

What are the consequences of UV-damage?

A
  • Adjacent pyrimidines covalently link through formation of a 4-member ring structure (CPD or thymine dimer)
  • Dimers will form between any 2 neighboring pyrimidine bases; helix bends 7-9o
  • Pyrimidine 6-4 Pyrimidone photoproducts (6-4PPs); helix bends 44o
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7
Q

What are some occupational exposures and their consequences?

A

-Diet high in salt & nitrate: stomach, esophagus

  • Diet high in fat, low in fiber, fried foods: bowel, pancreas, prostate, breast
  • Tobacco & alcohol: mouth, throat, lung, kidney, bladder
  • Asbestos: mesothelioma (damage to mesothelial cells in lining of chest & abdominal cavities)
  • Cigarette smoking: polycyclic aromatic HCs lead to G->T transversions in p53 tumor suppressor gene -> delayed lung cancer.
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8
Q

What are some carcinogens and their effects?

A

1) Arsenic: skin carcinoma, bladder cancer
2) Asbestos: mesothelioma
3) Benzene: acute leukemias
4) Radium: osteocarcinoma
5) Vinyl Chloride: liver angiosarcoma

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9
Q

What is the Ames test?

A

Test to determine the potential carcinogenic effects of chemicals:

1- Potential mutagen + histidine-dependent salmonella + homogenized liver extract are plated in agar w/o histidine

2- Incubation at 37C for 2 days 3- Count colonies of histidine-independent bacteria (these are the mutants)

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10
Q

What are some anticancer compounds that damage DNA?

A

Cisplatin, carboplatin, daunorubiicn, doxorubicin, oxaliplatin, and picoplatin

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11
Q

What are some cellular responses to DNA damage?

A

1- Checkpoint activation: signal transduction, transcriptional regulation, cell cycle arrest

2- DNA damage repair: excision repair, recombination prepair

3- Apoptosis

4- DNA damage tolerance: bypass polymerase

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12
Q

What are the 3 major types of DNA repair?

A

1- Direct reversal of damage (photolyase, AGT)

2- Excision repair (MMR, BER, NER)

3- Recombinatorial repair (DBS-repair)

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13
Q

What is direct reversal of damage?

A

A) Photolyase: reverses UV-induced damage in E.coli, plants, & some animals. Binds T-dimers & 6-4PP, absorbs a blue-light photon, & splits the photoproduct. Not in humans.

B) AGT (I-2 O6-akyltransferase, or MGMT): transfers the methyl group to a cysteine in the enzyme irreversibly (not in plants & S.pombe)

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14
Q

What are the types of excision repair?

A

A) Mismatch repair (MMR)

B) Base excision repair (BER): fixes deamination

C) Nucleotide excision repair (NER*): removes bulky adducts -UV damage

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15
Q

What is HNPCC?

A

-Hereditary nonpolyposis colorectal cancer (Lynch syndrome). Increases risk for colon cancer

  • Associated w_/Mut proteins_ from mismatch repair
  • 5% of colon cancers are the result of mutations in mismatch repair
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16
Q

What is the effect of O6-methyl-Guanine (nucleotide analog)?

A
  • It leads to cell arrest & apoptosis
  • MMR sees it as a pair mismatch, causing futile cycles of nt removal/synthesis causing breaks
  • If there is a high level of AGT, O6-meG can be quickly removed
  • Highly mutagenic
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17
Q

What is the mechanism of mismatch repair, MMR?

A

1) Mut proteins recognize mismatch
2) Identify methylated (parental) strand, and cleaves daughter strand
3) Segment is then filled by polymerase, & ligase joins to original strand.

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18
Q

What is the mechanism of base excision repair, BER ?

A

1) Deamination is identified (C-> U)
2) DNA-N-glycosylase generates AP site by hydrolysis of N-glycosidic bonds & removes base
3) Apyrimidinic (AP) endonuclease nicks the backbone at 5’ side of AP site generating a 3’-OH terminus
4) Cleavage of deoxyribose by lyase activity
5) DNA Pol/ ligase fill in and seal it.

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19
Q

What is the mechanism of photolyase?

A

1) Enzyme recognizes & binds to damage
2) light absorption by chromophore converts it to excited state
3) chromophore donates an electron to the cyclobutyl dimer
4) dimer is destabilized -> result in monomeric pyrimidines

20
Q

How can a defect in BER lead to cancer?

A
  • Mutation in DNA N-glycosylase MutY leads to APC mutation and colon cancer
  • It normally recognizes 8-OXO-G–A mispair.
21
Q

What is the mechanism of nucleotide excision repair, NER?

A

*Critical for repair of UV damage

  1. Damage recognition based on disrupted pairing by XPC or CSB/CSA-RNA pol holoenzyme
  2. Recruitment of proteins (TFIIH, XPG, XPA, XPB) to assemble repair complexes
  3. Double incisions remove 24-32 nt by XPG & XPF
  4. Excision of damage-containing nt’s
  5. Filling in and ligation
22
Q

What is Xeroderma Pigmentosum?

A
  • Deficient NER nucleotide excision repair disorder
  • Severe light sensitivity
  • Severe pigmentation irregularities
  • Frequent neurological defects
  • Early onset of skin cancer at high incidence & other cancers
23
Q

What diseasess are NER defecs linked to?

A
  • Xeroderma Pigmentosum
  • Cockayne’s syndrome (CSA/CSB)
  • Trichothiodystrophy (XPB and XPD)
24
Q

What is Cockayne’s syndrome?

A
  • Premature aging of some tissues, dwarfism, facial and limb abnormalities, neuronal abnormalities
  • Carry mutations in genes involved in or related to NER (XPB, XPD, XPG)
25
Q

What is Trichothiodystrophy?

A
  • Premature aging of some tissues, brittle hair, facial abnormalities, short stature, some light sensitivity
  • Mutations in genes involved in or related to NER (XPB, XPD)
26
Q

What is Recombinatorial repair, DBS (3)?

A
  • Mechanism to repair ds-breaks caused by: IR, ROS, meiosis, VDJ recombination
  • A) Homologous recombination (HR): between sequences that are nearly identical
  • B) Non-homologous end-joining (NHEJ) // Site Specific: between sequences with a limited stretch of similarity; involves specific sites.
27
Q

What is homologous recombination repair, HR ?

A

1) Enzyme recognizes break
2) End processing produces two ends; generation of ssDNA
3) Strand invasion -holiday junction formation -branch migration
4) Resolution of intermediates
5) ligation

*Use undamaged sister chromatids as template

28
Q

What is Site-specific / Non-homologous end-joining, NHEJ?

A
  • Moves specialized specialized sequences called mobile genetic elements (contains transposase) between non-homologous sites
  • Gene order can be altered, & new info added
  • Ex. Mating type switch in yeast & VDJ recombination for Ig diversity
  • STEPS: ** ** 1) Ku heterodimer binds to DNA ends, 2) DNA-activated kinase recruited, 3) DNA ends are processed, 4) Ligase 4 joins the ends
29
Q

How can DNA repair pathways be exploited for cancer therapy?

A

There are different repair pathways, but tumor cells tend to have a preferred pathway. If a drug then inhibits such pathway, cancer cells cannot be repaired and die. Normal cells have other repair pathways, so they can continue to live.

30
Q

What are the eukaryotic homologous recombination proteins?

A

-Initiation: end processing (Mre11/Rad50/ NBS1(XRS2)/RPA (ssDNAbp),

-Pre-synapsis: RPA, Rad51 (strand exchange), Ras 52/54/59, BRCA 1/2

-Heteroduplex extension: Rad51, Pol&, PolE

-Resolution: BLM helicase, Top3, Mus81 (endonuclease), EME1, XRCC3, Lig 1

-There might be crossing over

31
Q

What is meiotic recombination (HR) & what is the pathway?

A

The exchange of genetic materials between parents before giving it to the progeny:

1- Two homologous chromosomes are paired

2- One chromosome is cut

3- Exonuclease exposes ss 3’-end

4- Strand invasion joins chromosomes

32
Q

What are the DNA damage checkpoints actvated upon DNA damage?

A

-Block entry into S-phase through p53, Chk2, Cdc25

  • Slow DNA replication by inhibiting late origin firing through ATR
  • Block entry into M phase through ATR & Chk1
33
Q

How does checkpoint activation work?

A

1) Sensor proteins recognize damage (ATR-protein kinase, Rad17, ATM, Hus 1, ATRIP, etc.)
2) Mediators (protein complex assembly: BRCA1, Claspin, MDC1) signal to transducers
3) Chk1/2 transducers signal to effectors
4) Effector, p53 and Cdc25 phosphatase, down-regulate the cell cycle.

34
Q

What is the biological significance of recombination?

A
  • Generate genetic diversity
  • Repair of DNA breaks
  • Recombinational bypass of bulky DNA adducts
  • For chromosomal pairing during meiosis
35
Q

What happens when p53 is activated?

A

-p53 is acticvated by: hyperproliferative signals, DNA damage, telomere shortening, hypoxia

-Active p53 leads to cell cycle arrest, senescence, or apoptosis

36
Q

What are some examples of DNA damaging agents and their repair mechanism?

A

*BER: X-rays, O-radicals, alkylating agents, spontaneous reactions

*NER: UV light, polycyclic aromatic HCs

*MMR: replication errors

*HR/EJ: x-rays, anti-tumor agents

37
Q

What are some examples of DNA damage mechanisms?

A
  • Point mutations
  • Transition & transversions
  • Insertions & deletions
  • Expansion & contraction of microsatellite repeats
  • Chromosome rearrangements (translocations, inversions, deletions)
38
Q

What are microsatellites?

A
  • Tandem repeats composed of subunits, 1-6 nucleotides
  • Account for 3% of human genome
  • During replication, they change in length at a rapid rate
  • Polymorphic, so they are used for parentage & forensic analysis
  • Expansion in protein coding or 5’ UTRs can cause diseases (HD, FXS)
39
Q

What are the DNA mutations and their effects on polypeptide?

A
  • Missense: AA substitution
  • Neutral: change involved similar aa, no effect in function
  • Silent: different codon, but same aa
  • Nonsense: premature stop codon
  • Frame shift: functional effects
40
Q

What are some inherited syndromes associated with defects in DNA repair?

A

_-Bloom syndrom_e (cancer, stunted growth): accessory DNA helicase for replication

  • 46 BR patient (hypersensitivity to DNA-damaging agents): DNA ligase I
  • Werner syndrome: DNA helicase & 3’-exonuclease,
41
Q

What is Ataxia Telangiectasia?

A
  • Neurodegenerative & cancer-prone disorder
  • Progressive ataxia
  • Tumors of lymphoid origin, T-cell leukemias; impaired lymphocyte proliferation
  • Defective DBS response bc mutation in ATM, which interferes with NHEJ
42
Q

What is FANCD1?

A
  • Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome
  • Can cause aplastic anemia, GI problems, thumb and radial abnormalities, acute myeloid leukemia, short stature, mycrocephaly, head & neck, breast, ovarian, or prostate cancer
  • Due to hypomorphic mutation in BRCA2 & other genes, which i_nterferes with HR_
43
Q

What is ATR-Seckel syndrome?

A
  • Microcephalic dwarfism, developmental delay, dental malocclusion
  • Due to hypomorphic mutation in ATR gene, which impairs DBS repair
44
Q

What disorders are similar to AT caused by different gene mutations?

A
  • ATLD: hypomorphic mutation in MRE11
  • NBS: mutation in NBS1
  • FA: mutation BRCA2 & other genes
45
Q

What is RS-SCID & Lig4 syndrome?

A
  • Problems with VDJ recombination
  • RS-SCID: null-> severe combined immunodeficiency. Hypomorphic ->partial B/T cell deficiency. Due to a mutation in Artemis gene
  • Lig 4: due to hypomorphic mutation in LIG4 gene. Pancytopenia or combined immunodeficiency, developmental delay, lymphoid tumors, psoriasis, diabetes II, atypical bone maturation

BIOCHEMISTRY 2 (12 decks)

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