DNA damage and repair

Question 1

describe the structure of DNA bases and consequence of this *

Answer 1

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

Question 2

describe deamination *

Answer 2

bases are nitrogenous so contain amine groups

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

this occurs frequently, sometimes spontaneously

Question 3

examples of deamination *

Answer 3

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

Question 4

describe chemical modification *

Answer 4

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

Question 5

describe photodamage *

Answer 5

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

Question 6

what is a base pair mismatch *

Answer 6

mutated base with normal base on other chain

causes a bulge in the DNA

Question 7

how can you see a thiamine dimer in DNA template *

Answer 7

disrupt dna topology

= distortion in unit

Question 8

describe nicks *

Answer 8

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

Question 9

what can cause DNA damage *

Answer 9

chemicals - carcinogens

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

radiation

• ionising
• solar
• cosmic

Question 10

what is the principle of causing DNA damage in chemo *

Answer 10

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

Question 11

what is the importance of understanding DNA damage *

Answer 11

can lead to mutation = lead to cancer

chemo

Question 12

DNA damage that can be caused by carcinogens *

Answer 12

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

Question 13

describe the process of mammalian metabolism *

Answer 13

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

Question 14

describe polycyclic aromatic compounds *

Answer 14

they have double bonds so are reactive

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

known carcinogens

Question 15

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

Answer 15

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

Question 16

describe alfatoxin B1 *

Answer 16

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

Question 17

describe 2-napthylamine *

Answer 17

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

Question 18

describe UV radiation as a carcinogen *

Answer 18

causes thymine dimers - driver of skin cancer

Question 19

describe ionising radiation as a carcinogen *

Answer 19

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

Question 20

describe oxygen free radical attack on DNA *

Answer 20

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

Question 21

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

Answer 21

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

therefore the genome is kept in tact

Question 22

describe the role of p53 *

Answer 22

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

Question 23

what are the 4 types of DNA repair *

Answer 23

direct reversal of DNA damage

base excision repair - apurinic and apyrimidinic damage

nucleotide excision repain - for bulky DNA adducts

during or post replication repair

Question 24

describe direct DNA repair *

Answer 24

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

Question 25

describe DNA mismatch repair \*

Answer 25

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.

Question 26

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

Answer 26

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

Question 27

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

Answer 27

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

Question 28

what are human genetic diseases involving NER

Answer 28

xeroderma pigmentosum trichothiodystrophy cockayne's syndrome

Question 29

describe xeroderma pigmentosum \*

Answer 29

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

Question 30

describe trichothiodystrophy

Answer 30

sulfer deficient brittle hair facial abnormalities short stature ichthyosis - fish like scales on skin light sensitivity in some cases neurological

Question 31

describe cockayne's syndrome

Answer 31

dwarfism light sensitivity in some cases facial and limb abnormalities neurological abnormalities early death due to neurodegeneration

Question 32

describe repair of double strand breaks \*

Answer 32

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

Question 33

when are double strand breaks in DNA made \*

Answer 33

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

Question 34

3 fates of carcinogen damage leading to DNA mutations \*

Answer 34

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

Question 35

therapeutic agents that can cause DNA damage \*

Answer 35

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

Question 36

what is the sequence of carcinogen testing \*

Answer 36

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

Question 37

describe structural/SAR testing for carcinogens \*

Answer 37

by looking at structure, know whether it would be a carcinogen or not if it is likely to be, progress to bacterial gene assay

Question 38

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

Answer 38

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

Question 39

describe in vitro mammalian cell assays \*

Answer 39

test mammalian cells with chemical in presence of liver S9 look for chromosomal damage

Question 40

describe in vitro micronucleus assay \*

Answer 40

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)

Question 41

describe murine bone marrow micronucleus assay \*

Answer 41

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

Question 42

how do cells repair DNA damage \*

Answer 42

change gene expression = ativation of gene repair pathways