lecture 13- mechanisms of DNA repair Flashcards
the most important cell function is to ___, this is done by ___
preserve genetic material (keep DNA integrity)
done by mechanisms of DNA repair
name 6 mechanisms of DNA repair
mismatch repair
direct reversal of DNA damage
base excision repair (BER)
nucleotide excision repair (NER)
non-homologous end joining (NHEJ)
translesion DNA synthesis (TLS)
mechanisms of DNA repair are the most ___ mechanisms of the cell because they use a lot of ___
costly
energy repairing DNA
mismatch repair fixes ___
mismatched nucleotide replication errors
mismatch repair involves the removal of several nucleotides from which strand?
the new strand
briefly describe mismatch repair
usually “Mut” family enzymes- scan DNA, have to differentiate old parental DNA from new DNA, methylation– only parental (template for new DNA) is methylated, the enzyme knows the methylated enzyme is the correct one
- enzyme binds the mismatch, scans DNA bidirectionally, forms a loop…cleaves unmethylated DNA ahead of mismatch, helicase opens dsDNA and Pol I exonuclease degrades newly replicated DNA strand past the mismatch – Pol III fills gap – ligase seals DNA
direct reversal (repair) of DNA damage corrects ___
damaged nucleotide base in one step
name 2 examples of direct reversal of DNA damage
1- photoreactivation
2- removal of alkyl groups
describe direct reversal of DNA damage in photoreactivation
repairs cyclobutane pyrimidine dimers (which form when DNA is under UV light)
- enzyme responsible for separating the dimer is DNA photolyase
- photolyase presents 2 chromophore groups, absorbs a photon of visible light and passes excitation energy (electrons) to FADH
- chromophore = group that absorbs light (transfers electrons)
DNA photolyase uses the energy derived from ___ to reverse the damage caused by ___
absorbed visible light
UV light
describe direct reversal of DNA damage- removal of alkyl groups
repair of oxidized nucleotides
- O6 methylguanine is repaired by enzyme O6methylguanine-DNA methyltransferase to become guanine again by catalyzing the transfer of the methyl group to one of the enzymes own cysteine residues
- enzyme is degraded after (fixes only one DNA alteration)
- remember O6 methylguanine pairs with thymine (transition)
do photolyases repair pyrimidine dimers in humans?
no
Base Excision Repair functions at the level of ___ and is involved in several types of repair mechanisms
single damaged nucleotide
name 3 repair mechanisms by BER
1- removal of single damaged base (base was modified/damaged by alkylation, oxidation, or deamination); most uracils are removed by BER (deamination of cytosine forms uracils in DNA)
2- removal of abasic sugar (recognition and repair of AP sites- apurinic and apyrimidinic)
3- repairing single strand breaks that do not have the proper ends for ligase
what recognizes the damaged base in BER?
DNA glycosylase
describe BER in bacteria
formation of AP site — glycosylase cleaves base — AP endonuclease cleaves DNA and creates nick — 3’ OH and 5’ dRP — Pol I nick translation activity — ligase seals DNA
describe BER in eukaryotes
2 paths for BER (long and short path)
- DNA glycosylase — AP endonuclease forming nick — in long path: Polymerase without 5’-3’ activity, need Flap Endonuclease (about 10 nuc’s fixed)
. in short path: Polymerase beta removes sugar phosphate from AP site (only 1 damaged nucleotide is fixed)
BER in eukaryotes, ___ nuc’s are fixed in long patch and ___ in short patch
about 10
1
glycosylases scan DNA and ___ …
flip the damaged base out of the helix and remove it if it fits in the catalytic site
there are two main types of DNA glycosylases:
1- highly specific to a particular damaged base
2- recognizes oxidative damage in general (diverse substrates)
Nucleotide Excision Repair target what?
large, bulky lesions and removes DNA on either side of them
NER is the predominant repair pathway for removing _____
pyrimidine dimers, 6-4 photoproducts, and several other bulky base adducts, including benzo pyrene-guanine
describe process of NER
- protein complexes recognize a variety of base damages resulting in distortions to DNA structure
- NER enzymes cleave damaged DNA strand on both sides of the lesion (removing more than the damage itself)
- specialized helicase “releases” cut fragment
- DNA polymerase fills in the single stranded gap using undamaged strand as a template
- ligase closes the nick
unlike BER, NER does not…
require specific recognition of a damaged nucleotide
NER in E. coli (bacteria) involves primarily 4 proteins:
UrvA, UrvB, UrvC, UrvD
describe NER mechanism in bacteria
UrvA/UrvB scan for distortion in DNA –> UrvA then leaves, UrvB melts short stretch of DNA –> UrvC nicks both sides of distortion (about 12-13 bases) (in BER, 1 nick ; in NER, 2 nicks- 3’ and 5’) (UrvC called an exinuclease because it makes 2 nicks) –> UrvD (helicase- breaks H bonds) releases the fragment of DNA damage –> DNA Pol I fills gap, ligase closes gap
NER in bacteria: function of UrvA
recognizes lesion
NER in bacteria: function of UrvB
unwinds DNA
NER in bacteria: function of UrvC
exinuclease- nicks on both sides
NER in bacteria: function of UrvD
helicase- releases fragment of DNA damage
NER in bacteria: function of Pol I
fills in gap
NER in bacteria: function of ligase
seals DNA
which repair mechanism fixes pyrimidine dimers in humans?
NER
most of what we know about NER fixing pyrimidine dimers in humans is from research done on…
Xeroderma Pigmentosum- individuals extremely sensitive to sunlight
- mutations in at least seven genes (XPA-XPG) result in XP (defective NER)
describe NER mechanism in eukaryotes
XPC recognizes lesion and recruits XPB & XPD (unwind DNA) –> they recruit XPF & XPG (XPF creates nick in 5’, XPG creates nick in 3’) –> RPA stabilizes the bubble –> PCNA is clamp loader that recruits poly’s to the rregion that fill gap –> ligase seals DNA
NER mechanism in eukaryotes has global pathway, but also…
Transcription-coupled repair
- specifically targets repair to actively transcribed DNA
- when DNA being transcribed to RNA, RNA polymerase recognizes the damage and stalls transcription (RNA pol contains XPB & XPD) –> then continues on to XPF & XPG…
double-strand breaks can be repaired by ___
homologous recombination (error “free”) typical route
a repair mechanism for double strand breaks for phases of cell cycle when so sister chromatids are present is ___
non-homologous end joining (NHEJ)
NHEJ usually leads to ___
mutations- loss of DNA
briefly describe NHEJ
NHEJ present in all eukaryotes and some bacteria
- Ku70 & Ku80 bind to DNA ends in double strand breaks, they recruit other enzymes (DNA-PKcs and Artemis) - this complex fixes the double strand break
- XLF, XRCC4, and ligase IV seals the gap
translesion DNA synthesis is repair for…
lesion at a replication fork after DNA strands have been unwound
- damaged DNA encountered at fork
describe translesion DNA synthesis
DNA polymerase IV and V are used to extend DNA strand along template lesion —> DNA poly III comes again to continue replication
