what is the estimated daily incidence of DNA damage?
10,000 damage events occur to cells each day!
what are the greatest contributors to chromosomal DNA damage
intracellular rxns of hydrolysis, methylation, reactive oxygen species (ROS)
also UV light exposure to skin cells = DMA damaging
why would a replication error occur?
what type of damage does it cause?
b/c DNA polymerase is very processive, it's working fast/a lot, so might place an incorrect nucleotide and thus cause a mismatched base pair
what are the repair processes to fix mismatched bases?
1) DNA polymerase delta/epsilon have proofreading ability
2) mistmatch repair system if proofreading doesn't work
how do DNA pols delta/epsilon do proofreading to rectify a mismatched base?
DNA pol delta and epsilon have intrinsic 3' to 5' exonuclease activity that cuts out mismatched base that was inserted, inserts correct ntd before continuing synthesis
if DNA pol delta/epsilon's proofreading doesn't work, what is the result in the DNA?
mismatch causes downstream problems b/c new synthesized DNA has both wildtype and mismatched nucleotide which gets coded into DNA and would -> txn -> tln -> incorrect protein expression or none
when does the mismatch repair system kick in? how does the mismatch repair system work?
if DNA pol delta/epsil's proofreading doesn't work, MMR kicks in
MMR=enzymes/protein complexes (MSH, MLH) patrolling the DNA, looking for the mismatch
MMR system binds to the mismatch, recognize the 3' end of a nick of newly synthesized DNA, degrade DNA stretch containing the mismatch, make a gap where mismatch was
then repair single stranded DNA w/ DNA pol Delta
what does an inherited mutation in mismatch repair result in?
susceptibility to colon cancer, specifically HNPCC, hereditary non-polypopst colon cancer, cause 15% of all colon cancers in the USA
more generally, repair system defect means mistakes don't get fixed and can get end up w/ mutation in a tumor suppressor gene so cell can divide out of control!
what are MSH and MLH?
proteins of mismatch repair system
what causes HNPCC?
inherited mutation in 1 copy of the MLH gene
therefore have a defective copy of an important protein in the mismatch repair system
mutations can accumulate and result in tumor
what kind of damage do chemicals, smoke, and ROS, as well as problems w/ DNA, cause?
how is it dealt w/?
1) base excision repair
2) direct repair
what does "modified base" damage mean?
modified bases can pair w/ the wrong base
upon DNA replication, gets fixed into the genome
caused by chemical exposure that causes purine/pyrimidine bases to be damaged
what is the enzyme for base excision repair?
glycosylase = enzyme that recognizes incorrect base, clips it out
eg = uracil glycosylase
how does base excision repair work?
1) DNA glycosylase patrols genome, looking for modified bases
2) glycosylase cuts bond between base and 1'C of ribose sugar, creates an abasic site
AP endonuclease creates nick in phosphate-sugar backbone at that site
3) this abasic site is recognized by other enzymes
deoxyribosephosphodiesterase removes abasic phosphate-sugar group
4) DNA polymerase beta fills in the correct missing ntd
5) ligase reseals the phosphate-sugar backbone
what is "direct repair"? what does it fix?
2nd method of fixing modified base
MGMT, methylguanosine methyltransferase = direct reversal protein
works on bases that have been methylated improperly
MGMT binds to improperly methylated base, transfers the methyl group to a Cys residue in its active site, returns base to its active state
what kind of damage does UV light cause?
what and how does a pyrmidine dimer form?
UV light causes neighboring pyrimidine bases covalently bond w/ each other
sunlight can damage ntds, gives nearby adjacent bases covalent bonding
causes double stranded helix to pucker due to Thymine-Thymine dimer, for ex
wrecks DNA structure b/c RNA pol and DNA pol cannot get through
what is cisplatin? what does it do?
cancer drug that causes thymine dimers, overwhelms the pyrimidine dimer repair system
what are the repair processes to rectify pyrimidine dimers?
1) nucleotide excision repair (NER)
2) translesion synthesis
how does nucleotide excision repair (NER) work?
1) XP protein (xeroperma pigmentosum) recognizes the distorded DNA region
2) other XP proteins unwind and excise a patch of DNA containing lesion
3) DNA Pol Epsilon or Delta fills in
4) DNA ligase seals phosphodiester bond
how does translesion synthesis rectify pyrimidine dimers?
insertion of a new base opposite the site of stalled DNA replication per pyrimidine dimer
done by unique DNA polymerases (H, i, zeta) that're not processive and error-prone, but can replicate past pyrimidine dimer
they put in a new base opposite the dimer, restoring ability of DNA replication
although might insert incorrect base, this is much better than having total block to replication in absence of repair/translesion synthesis
what kidn of damage does ionizing radiation (X-Ray) exposure cause?
double stranded DNA break
why is a double stranded DNA break a problem?
is incompatible w/ cell's mechanism of equally distributing chromsomes to daughter cells
cannot repair based on complementary strand, in this case
how are double stranded DNA breaks rectified?
1) non-homologous end joining (NHEJ)
2) homology directed repair
how does non-homologous end joining rectify double stranded DNA breaks?
imprecisely ligates broken ends
via this method, you introduce an error; but repairs DNA so cycle can continue
how does homology directed repair rectify double stranded DNA breaks?
uses other chromosome as basis for repair of broken chromosome
BRCA2 protein binds and recruits Rad51
Rad51 is radiation-sensitive; coats strand & then allows single stranded coded piece of DNA to interact w/ the other, undamaged chromatid
describe process of non-homolgous end joining
to repair break in double stranded DNA
1) Ku proteins bind to 2 ends of break, form a synapse
2) helicase activity of Ku unwinds both ends
3) short single-stranded region in one DNA base-pairs w/ homolgous region in the other
forms microhomology region
5) unpaired 5/ regions are removed, free ends are ligated
In an individual with an inherited mutant copy of a DNA repair gene, a cell that suffers a somatic mutation in the second copy of the gene can result in drastic consequences to the individual. On the other hand, in an individual with an inherited mutant copy of a metabolic enzyme gene, a cell that suffers a somatic mutation in the second copy of the gene will be of no consequence to the individual. Explain.
DNA repair genes are crucial to ensuring individual can have correct DNA sequences
However metabolic enzyme genes wouldn’t cause individual such suffering because you can replace whatever that metabolic enzyme gene isn’t doing w/ therapy
Whereas if have damage to DNA repair gene, get more widespread incorporation of incorrect sequences in genome, this causes widespread problems
Why are xeroderma pigmentosum patients more susceptible to skin cancers than all other cancers?
These individuals lose their ability to do nucleotide excision repair
If an individual is exposed to UV light, usually the resultant pyrimidine dimers can be rectified via nucleotide excision repair or translesion synthesis, but nucleotide excision repair is more common method of fixing this
Pyrimidine dimerization causes DNA double stranded helix to pucker, wrecking DNA struck and blocking RNA and DNA polymerase from getting through and doing replication
Loss of ability to do DNA replication à skin cancer
What is the advantage that DNA polymerases involved in translesion synthesis are not highly processive?
DNA polymerases involved in translesion synthesis not being processive means that they can replicate past the pyrimidine dimer and put in a correct/incorrect base opposite the dimer, then continuing w/ DNA replication which had been blocked
Other DNA polymerases can not do this
Their lack of processivity means they will add that base in the position opposite the dimer and fall off some time soon, which is good since they often insert incorrect bases when they are working to fill in opposite of dimers – we wouldn’t want a polymerase that inserts incorrect bases to be highly processive, we want it to do the work it must do and then fall off of the DNA and stop replicating (potentially) incorrectly