LEC30: DNA Repair Flashcards Preview

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Flashcards in LEC30: DNA Repair Deck (30):

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 

ligates together 


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/?

modified base

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?

pyrimidine dimer


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?

advantages, disadvantages?

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


A image thumb

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


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