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cancer - year 2 > DNA damage and repair > Flashcards

DNA damage and repair Flashcards

1
Q

describe the structure of DNA bases and consequence of this *

A

planar carbon rings therefore can be activated chemically and so are able to react with other molecules

double bond can be activated

rings allow delcalisation of electrons

eg structural similarly so remove methyl group of thiamine means you get uracil

this results in DNA changes

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2
Q

describe deamination *

A

bases are nitrogenous so contain amine groups

primary amino groups of bases are unstable - can be converted to ketogroups

this occurs frequently, sometimes spontaneously

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3
Q

examples of deamination *

A

NH3 of cytosine is removed and released = uracil which = thiamine, therefore deamination converts cytosine to thiamine

adenonine to hypocanthine

guanine to xanthine

5-methyl cytosine to thymine directly

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4
Q

describe chemical modification *

A

oxidation reactons - double bonds are broken so they can react

hyper-reactive oxygen (eg singlet ox, peroxide radicals, hydrogen peroxide and hydroxyl radicals are generated as byproducts during oxidative metabolism or by ionising radiation (x/gamma rays)

these modify DNA bases

eg thiamine can be oxidised = thiamine glycol, thiamine’s double bonds open and the OH groups formed are chemically reactive - they react with chemical reactive species that favour reactive bases - these can be larger molecules that are carcinogenic ie thiamine glycol covalently links to other species forming adducts

environmental chemicals including natural ones eg in food can modify bases by methylation/alkylation, normal met can cause alkylation

addition of larger molecules defines adducts

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5
Q

describe photodamage *

A

due to UV light - occurs in skin

UV light absorbed by nucleic acid bases nad resulting influx of energy causes chem changes

UV activates the thiamine ring = thiamine dimer

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6
Q

what is a base pair mismatch *

A

mutated base with normal base on other chain

causes a bulge in the DNA

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7
Q

how can you see a thiamine dimer in DNA template *

A

disrupt dna topology

= distortion in unit

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8
Q

describe nicks *

A

because of reative particles and high energy radiaton- including high energy x-ray and cosmic rays

causes a break in phosphodiester backbone - this is a nick

if get lots of nicks together = gap = single stranded DNA

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9
Q

what can cause DNA damage *

A

chemicals - carcinogens

  • diet - result of metabolism
  • lifestyle
  • env
  • occupational
  • medical
  • endogenous - from normal metabolism

radiation

  • ionising
  • solar
  • cosmic
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10
Q

what is the principle of causing DNA damage in chemo *

A

cause damage that cells cant recover from - overwhelm cell with DNA damage = apoptosis

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11
Q

what is the importance of understanding DNA damage *

A

can lead to mutation = lead to cancer

chemo

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12
Q

DNA damage that can be caused by carcinogens *

A

base dimers and chemical cross links

double and single strand breaks (nicks)

dna adducts and alkylation

base hydroxylation and abasic sites - bases hydroxylated by reactive oxygen species = base change/destroyed to extent that it is no longer a base = abasic site, however the underlying DNA is in tact. clearly damaged because have base on 1 strand but not on other This is the predominant type of damage seen in active cells, occurs because of metabolism

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13
Q

describe the process of mammalian metabolism *

A

phase 1

  • addiction of func gps eg oxidation, reduction, hydrolysis
  • mediated by cytochrome p450
  • convert hydrophobic carbon ring into something polar and sol so it can be excreted - make them available for phase 2

phase 2

  • conjugation of phase 1 func groups
  • eg sulphation, glucuronidation, acetylation, methylation, aa and glutathione conjugation
  • generates water sol metabolites
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14
Q

describe polycyclic aromatic compounds *

A

they have double bonds so are reactive

common env pollutants, from combustion of fossil fuels, and tabacco

known carcinogens

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15
Q

describe benzo[a]pyrene (B[a]P) *

A

ubiquitous - in coal tar, tabacco and grilled meat

gp 1 carcinogen - potent

B[a]P not capable of DNA damage alone

it is metabolised by p450 enzymes including epoxide hydrolase (EH)

this forms epoxide form of B[a]P which reacts with DNA to form adducts = is a carcinogen - it attaches to bases that have been chemically activated and causes mutations

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16
Q

describe alfatoxin B1 *

A

potent human liver carcinogen

alone it is not a carcinogen, then is metabolised by cP450 enzymes and forms an epoxide

this reacts with DNA guanines at N7 position = adduct forms adducts at guanine

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17
Q

describe 2-napthylamine *

A

is oxidised by P450

in phase 2 met joins to glucuronyle to make soluble - ends up in urine

in the pH of urine is broken down into a chemically reactive nitrenium ion = reacts with DNA = bladder cancer

18
Q

describe UV radiation as a carcinogen *

A

causes thymine dimers - driver of skin cancer

19
Q

describe ionising radiation as a carcinogen *

A

generates free radicals in a cell

cause activation of DNA bases = chemical reactivity = nicks at extreme end by breaking phosphodiester bonds

includes oxygen free radicals - super oxide radica O2 rad, hydroxyl rad (HO rad)

possess unpaired electrons - electrophilic so seek out electron rich DNA

some act directly interacting with base structures, and some indirectly by formation of radicals as they pas through the cells

20
Q

describe oxygen free radical attack on DNA *

A

come about because of metabolism and generation of oxygen free radicals

double and single strand breaks

causes apurinic and apyrimidinic sites

cause base modifications

  • thiamine and cytosine glycols
  • ring opened of guanine and adensine
  • purines are converted into 8-hydroxypurines - these are chemically reactive and mutagenic - can undergo adduct formation
21
Q

what can you tell by the rate of dna repair and damage *

A

cells have capacity to cope because rate of repair is more than damage

therefore the genome is kept in tact

22
Q

describe the role of p53 *

A

it changes DNA expression = activation of a repair pathway

responds to a variety of insults eg mitotic apparatus dysfunction, dna replication stress and double starnd breaks

it is kept inactive by MDM2 - in activation MDM2 is lost

p53 is a transcription factor - activates pathways and genes that respond to the relevant signal ie DNA repair pathways or apoptosis if damage is extreme and cells cant be repaired

23
Q

what are the 4 types of DNA repair *

A

direct reversal of DNA damage

base excision repair - apurinic and apyrimidinic damage

nucleotide excision repain - for bulky DNA adducts

during or post replication repair

24
Q

describe direct DNA repair *

A

involves removal or reversal of damage by use of proteins which carry out specific enzymatic reactions

photolyses fix thiamine dimers are activated by light and recognise dimers, remove the dimers and allow repair by dna polymerase

O6 methylguanine-DNA methyltransferase (MGMT) reverses simple alkylation products - recognise base by methyl sites, take away the methyl group, base left in normal state then MGMT is degraded

alkyltransfases remove alkyl groups from bases

25
Q

describe DNA mismatch repair *

A

correct errors from DNA replication

involves scruitinisation of DNA for apposed bases that dont pair properly

mismatches are corrected by comparing old and new strands - proof reading

repair whne bulge in DNA becasue DNA pol has put in the wrong base - MSH and LSH proteins recognise the bulge

they bind around the mismatch, identify it

nuclease removes the base and the bases around it

dna polymerase repairs it

can only occur in DNA replication

preference for newly synthesised strand

Other systems deal with mismatches generated by base conversions, such as those which result from deamination.

26
Q

describe excision repair of DNA damage - base excision repair pathway *

A

removes small adducts

the base is damaged, but not the rest of the backbone

DNA glycosylase remove the base but doesnt effect the backbone - end up with abasic

AP-endonuclease cuts open the DNA in the strand and DNA polymerase adds the correct base

DNA ligase closes teh gap

27
Q

describe excision repair of DNA damage - nucleotide excision pathway (NER) *

A

repair DNA when base has mutated which has caused additional damage to DNA/ removal of a big adduct group

endonuclease makes nicks

helicase removes patch of DNA, including backbone

dna polymerase adds new bases and ligases fixes backbone

28
Q

what are human genetic diseases involving NER

A

xeroderma pigmentosum

trichothiodystrophy

cockayne’s syndrome

29
Q

describe xeroderma pigmentosum *

A

severe light sensitivity

severe pigmentation irregularities

early onset of skin cancer at high incidence, melamomas and eye tumours

frequent neurological defects

GIT cancer

several gene defects give the same phenotype

not curable - needs to be managed, eg by shifting day so that you function at night to avoid the sun

30
Q

describe trichothiodystrophy

A

sulfer deficient brittle hair

facial abnormalities

short stature

ichthyosis - fish like scales on skin

light sensitivity in some cases

neurological

31
Q

describe cockayne’s syndrome

A

dwarfism

light sensitivity in some cases

facial and limb abnormalities

neurological abnormalities

early death due to neurodegeneration

32
Q

describe repair of double strand breaks *

A

an exonuclease chews back the ends of the DNA to expose more of the chain in attempt to reveal bits that match to the other fragments

the fragments are then joined by transient base pairing of several nucleotides, folled by dna polymerisation, nucleolytic processing and ligation

in extreme case join bits that weren’t connected before - non-homolous end-joining - the free ends of DNA are picked up by Ku proteins - they hold DNA together and force the fragments to join

33
Q

when are double strand breaks in DNA made *

A

formation of Ab binding site genes - hypermutation of variable region of Ab - joining of segments

under physiological conditions during somatic recombination and transposition eg V(D)J recombination

during homolgous recombination in meosis

becasue of ionising radiation and oxidative stress induced DNA damage

34
Q

3 fates of carcinogen damage leading to DNA mutations *

A
  1. efficient repair - normal cell
  2. incorrect repair/altered primary sequence = dna replication and cell division has mixed mutations - affect ability to survive, replicate and grow - affects transcription/translation = aberrent proteins, and causes carcinogeneisis of critical targets are mutated - onchogenes/tumour suppressor genes
  3. if cell cant be recovered = apoptosis
35
Q

therapeutic agents that can cause DNA damage *

A

alkylating agents

agents that make bulky adducts eg cisplatin

agents that induce double strand breaks so start joining bits of DNA that shouldnt go together = corrupt genome = apoptosis - ionising radiation (radiotherapy), bleomycin, neocarzinostatin

36
Q

what is the sequence of carcinogen testing *

A

structural alerts/SAR

in vitro bacterial gene mutation assay eg Ames test with S. typhimurium

in vitro mammalian cell assay eg chromosome aberration, TK mutation in mouse lymphoma cell micronucleus assay

in vivo mammalian assay - eg bone marrow micronucleuys test transgenic rodent assay

investigate in vivo mammalian assays

37
Q

describe structural/SAR testing for carcinogens *

A

by looking at structure, know whether it would be a carcinogen or not

if it is likely to be, progress to bacterial gene assay

38
Q

describe bacterial gene assays to test whether something is a carcinogen *

A

Ames test

add chemical to be tested and rat liver enzyme preparation (have all essential enzymes for phase 1 and 2 met) to bacterior unable to synthesis histidine (eg salmonella strain) - this is a mutation, bacteria need histidine to live

if the chemical is a carcinogen it will mutate bacterial DNA = allow bacteria to make histidine

when chemical is carcinogen there is a higher number of bacterial colonies - can quantify by counting colonies

39
Q

describe in vitro mammalian cell assays *

A

test mammalian cells with chemical in presence of liver S9

look for chromosomal damage

40
Q

describe in vitro micronucleus assay *

A

cells treated with chemical and allowed to divide

binucleate cells assessed for the presence of micronuclei - if DNA very damaged it is bulged off as micronuclei especially in dividing cells

see them using staing that makes DNA fluresce

can stain the kinetochore proteins to see if chemical treatment caused clastigenicty (chromosmal breakage) or aneuploidy (chromosomal loss)

41
Q

describe murine bone marrow micronucleus assay *

A

treat animals with chemicals and examine marrow or peripheral erythrocytes for micronuclei

see how many nuclei in polychromatic red cells - shouldnt have any because the nucleocyte is lost

this is a direct measure of new carcinogen

42
Q

how do cells repair DNA damage *

A

change gene expression = ativation of gene repair pathways

cancer - year 2 (18 decks)

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