DNA damage and repair Flashcards
describe the structure of DNA bases and consequence of this *
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
describe deamination *
bases are nitrogenous so contain amine groups
primary amino groups of bases are unstable - can be converted to ketogroups
this occurs frequently, sometimes spontaneously
examples of deamination *
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
describe chemical modification *
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
describe photodamage *
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
what is a base pair mismatch *
mutated base with normal base on other chain
causes a bulge in the DNA
how can you see a thiamine dimer in DNA template *
disrupt dna topology
= distortion in unit
describe nicks *
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
what can cause DNA damage *
chemicals - carcinogens
- diet - result of metabolism
- lifestyle
- env
- occupational
- medical
- endogenous - from normal metabolism
radiation
- ionising
- solar
- cosmic
what is the principle of causing DNA damage in chemo *
cause damage that cells cant recover from - overwhelm cell with DNA damage = apoptosis
what is the importance of understanding DNA damage *
can lead to mutation = lead to cancer
chemo
DNA damage that can be caused by carcinogens *
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
describe the process of mammalian metabolism *
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
describe polycyclic aromatic compounds *
they have double bonds so are reactive
common env pollutants, from combustion of fossil fuels, and tabacco
known carcinogens
describe benzo[a]pyrene (B[a]P) *
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
describe alfatoxin B1 *
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
describe 2-napthylamine *
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
describe UV radiation as a carcinogen *
causes thymine dimers - driver of skin cancer
describe ionising radiation as a carcinogen *
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
describe oxygen free radical attack on DNA *
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
what can you tell by the rate of dna repair and damage *
cells have capacity to cope because rate of repair is more than damage
therefore the genome is kept in tact
describe the role of p53 *
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
what are the 4 types of DNA repair *
direct reversal of DNA damage
base excision repair - apurinic and apyrimidinic damage
nucleotide excision repain - for bulky DNA adducts
during or post replication repair
describe direct DNA repair *
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
describe DNA mismatch repair *
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.
describe excision repair of DNA damage - base excision repair pathway *
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
describe excision repair of DNA damage - nucleotide excision pathway (NER) *
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
what are human genetic diseases involving NER
xeroderma pigmentosum
trichothiodystrophy
cockayne’s syndrome
describe xeroderma pigmentosum *
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
describe trichothiodystrophy
sulfer deficient brittle hair
facial abnormalities
short stature
ichthyosis - fish like scales on skin
light sensitivity in some cases
neurological
describe cockayne’s syndrome
dwarfism
light sensitivity in some cases
facial and limb abnormalities
neurological abnormalities
early death due to neurodegeneration
describe repair of double strand breaks *
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
when are double strand breaks in DNA made *
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
3 fates of carcinogen damage leading to DNA mutations *
- efficient repair - normal cell
- 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
- if cell cant be recovered = apoptosis
therapeutic agents that can cause DNA damage *
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
what is the sequence of carcinogen testing *
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
describe structural/SAR testing for carcinogens *
by looking at structure, know whether it would be a carcinogen or not
if it is likely to be, progress to bacterial gene assay
describe bacterial gene assays to test whether something is a carcinogen *
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
describe in vitro mammalian cell assays *
test mammalian cells with chemical in presence of liver S9
look for chromosomal damage
describe in vitro micronucleus assay *
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)
describe murine bone marrow micronucleus assay *
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
how do cells repair DNA damage *
change gene expression = ativation of gene repair pathways
