LEC30: DNA Repair Flashcards
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
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
